Histopathological features in advanced abdominal pregnancies co-infected with SARS-CoV-2 and HIV-1 infections: A case evaluation.

Objectives: This study aims to provide a semi-qualitative histopathological report of the dual SARS-CoV-2 and HIV infected placentae in the third trimester of Advanced Abdominal Pregnancy (AAP). Study design: Four AAP placentae in the third trimester of pregnancy (two positive for HIV-1 and two positives for SARS-CoV-2) were histologically examined.
Results: The SARS-CoV-2+ HIV+ placentae were dysmorphic in shape compared to the flattened disc-like shape noted in the SARS-CoV-2+HIV-, SARS-CoV-2-HIV+and SARS-CoV-2-HIV- placentae. Diffused syncytial knots and syncytial degeneration were observed in all placentae. Intermittent cytotrophoblast increase, perivillous and intravillous fibrin deposition, mononuclear inflammatory cells with widespread degeneration/necrosis of the syncytiotrophoblast and microcalcification were pronounced in the SARS-CoV-2+HIV+ compared to the SARS-CoV-2+HIV- placentae. Vascular pathological changes included thrombi, ectasis, mural hypertrophy and atherotic vessels.
Conclusion: Elevated syncytial trophoblast injury, villitis, microcalcifications and mineralisation of the syncytial basement membrane in the AAP placentae may be due to SARS-CoV-2 viral transgression instead of HIV infection alone. Vascular malperfusion is suggestive of a hypoxic insult arising from a compensatory response to meet the fetal oxygen and nutrient demands of an AAP. Placentae from HIV infected women on antiretroviral treatment were characterised by vascular malperfusion.

Introduction

The province of KwaZulu-Natal, South Africa (SA) is considered the epicentre of the HIV pandemic where one in three pregnant women attending antenatal clinics are HIV infected [1], [2]. Human Immunodeficiency Virus infection in pregnancy increases the risk of maternal and perinatal morbidity and mortality [3]. Recently, the COVID-19 pandemic has been reported to increase the maternal mortality ratio in SA by 30% [4]. Similarly, a large multinational cohort study across 18 countries has demonstrated that COVID-19 in pregnancy is associated with increase in maternal and neonatal morbidity and mortality [5].

Advanced abdominal pregnancy (AAP) refers to a pregnancy that develops in the abdominal cavity and proceeds beyond 20 weeks of gestation [6], [7] and it is rare for these pregnancies to reach the third trimester with a healthy viable foetus [8]. To-date, management of the placenta at delivery is controversial. Ligation of the umbilical cord and leaving the placenta in situ is preferred by most surgeons because of the risk of maternal haemorrhage following placental removal. It is therefore not surprising that there is limited information on the histological evaluation of AAP placentae.

Histopathologic examination of placental tissue mimics a window to the health of both mother and baby. There is however a paucity of information on placental histology of AAP with viral infections. Hence, in the face of HIV and COVID-19 infections, we report on placental histopathology of four rare cases of AAP with live births.

Material and methods

Ethics approval

This study was conducted at a tertiary hospital in SA. Participants were referred from surrounding healthcare facilities for specialist care. Institutional ethical approval (BE026/010), regulatory health authority permission and informed consent were obtained prior to collection of placental samples.

Study population

The placentae were obtained from four women who had scheduled laparotomy to deliver the AAP with live foetuses in the third trimester of pregnancy. Of the four placentae, two were SARS-CoV-2 positive and two were SARS-CoV-2 negative women. The placentae were further stratified by the participants HIV status into SARS-CoV-2+ HIV+; SARS-CoV-2+ HIV-; SARS-CoV-2- HIV+ and SARS-CoV-2- HIV- sub-groups. The diagnosis of SARS-CoV-2 was confirmed with polymerase chain testing.

Placental handling during surgical removal

Laparotomy were performed through a midline abdominal incision. The placental location and findings at laparotomy are shown in Table 1. In all the cases, the omentum and bowel were closely adherent to the amniotic sac. The foetus was delivered after opening the amniotic sac furthest away from the placental attachment, at the least vascular site. Following delivery of the foetus, the umbilical cord was ligated, and a careful inspection was performed to identify the vascular supply to the placenta. The placenta was subsequently carefully mobilized, with the omentum and bowel separated from the sac. A systematic approach using surgical clamps and ties with ligation of the feeding vessels was performed. In all the cases, the infundibular pelvic and omental vessels were the main vascular supply. The placenta was removed “enbloc” and immediately stored in formalin for histopathological analyses. Placental weights were only available for the SARS-CoV-2+ infected placentae.

Table 1

Placental and Fetal Findings at Laparotomy.

Findings at surgery	SARS-CoV-2+ HIV+	SARS-CoV-2+ HIV-	SARS-CoV-2- HIV+	SARS-CoV-2- HIV-
Placental localization	Attached to the fundus of the uterus and right adnexa	Left adnexa and left upper quadrant	Superior to the uterus, left paracolic and left adnexa	Attached to right broad ligament and adnexa of uterus
Placental vascular supply	Right infundibular pelvic vessels	Mesentery of the transverse colon, left infundibular pelvic and retroperitoneal vessels in the region of the aorta	Left infundibular pelivic artery	Right infundibular pelivic artery
Placental weight (grams)	811	677	Not available	Not available

Histological placental preparation

Placental samples (central region) were fixed in 10% buffered formaldehyde, dehydrated, infiltrated and embedded into paraffin wax blocks as per standard laboratory procedure [9]. Sections of placental tissue (3 µm) were de-paraffinized, rehydrated for haematoxylin and eosin staining, dehydrated and “cover-slipped” as previously reported [10]. Sections were then examined using the ZEISS Axio Imager 2 and images were captured using the Zen (Blue edition) software (Cariss-Strasse, Oberkochen, Germany).

Results

The demographic and clinical data for all four groups are shown in Table 2. All participants were in their 3rd trimester of pregnancy, and one patient had a history of a previous ectopic pregnancy. None of the study patients had evidence of urinary tract infections or proteinuria. Blood pressure levels were normal in all participants, with no clinical, X-Ray or laboratory evidence of TB and syphilis co-infections. Also, two women were SARS-CoV-2 positive (one HIV+ and one HIV-) on PCR testing whilst two were SARS-CoV-2 negative (one HIV+ and one HIV-). Both SARS-CoV-2 positive patients were asymptomatic, diagnosed in the third trimester and delivery effected greater than 14 days from diagnosis. All babies at birth were alive, did not require neonatal intensive care and were alive at the time of hospital discharge.

Table 2

Clinical Characteristics and Laboratory Data of the Advanced Abdominal Pregnancies and their Babies.

Clinical Parameters	SARS-CoV-2+ HIV+	SARS-CoV-2+ HIV-	SARS-CoV-2- HIV+	SARS-CoV-2- HIV-
Maternal age (years)	36	21	39	29
Parity	0	0 + (1 Ectopic)	3	2
Gravidity	1	2	4	3
Blood Pressure (mmHg)	110/53	101/62	100/60	120/70
Gestational age (weeks)	34	37	36	30
Hemoglobin (g/dl)	13.8	13.3	11.1	10.3
Viral load (cells/ µL)	LTDL (FDC)	–	LTDL (FDC)	–
CD4 count (cells/µL)	500	–	484	–
TB screening	Negative	Negative	Negative	Negative
COVID screening	Positive (27 weeks)	Positive (34 weeks)	Negative	Negative
Rapid Plasma Reagin	Negative	Negative	Negative	Negative
Rhesus Grouping	Positive	Positive	Positive	Positive
Baby weight (kg)	1.98	2.95	2.78	1.2
Fetal abnormality	dysmorphic features with left foot abnormality	nil	nil	nil

LTDL: lower than detectable load; FDC: fixed dose combination

Placental findings

Gross morphological features

Macroscopically, the SARS-CoV-2+ HIV+ placentae was dysmorphic in appearance (Fig. 1a; approximately 811 g) vs the SARS-CoV-2+ HIV- placenta (Fig. 1b, 677 g). In contrast, the SARS-CoV-2- HIV- and SARS-CoV-2- HIV+ placentae were disc like in appearance. There was no evidence of infarcts. Apart from the umbilical cord insertion, differentiation between foetal (chorionic plate) and maternal portion (basal plate decidua) was difficult. The insertion sites of the umbilical cord were eccentric across all placentae.

Fig. 1

Advanced abdominal pregnancy: Macroscopic lateral view of (a) SARS-CoV-2+HIV+ and (b) superior view of SARS-CoV-2+HIV- placentae. Histopathological features of AAP SARS-CoV-2+HIV+ infected placental villi showing (c-d) mineralization of the basement membrane and (e-f) microcalcification (x40).

Histopathological features

The histopathological characteristics of all four AAPs are shown in Table 3. Foetal membranes for all placentae consisted of simple cuboidal epithelium resting on a thin basement membrane with underlying thin band of loose connective tissue. Dystrophic calcifications were observed in the decidua, septa and basal plate. Structurally, placental villi lying within the intervillous space were covered by a syncytiotrophoblast layer regardless of the infection type. The syncytiotrophoblast layer was made up of a multinucleated syncytium with eosinophilic cytoplasm, multiple intracytoplasmic vacuoles and pyknotic nuclei. Diffused multifocal syncytial knots were present in all placentae.

Table 3

Histopathological evaluation of the placenta.

Histopathologic features	SARS-CoV-2+ HIV+	SARS-CoV-2+ HIV-	SARS-CoV-2- HIV+	SARS-CoV-2- HIV-
Mineralization of basement membrane	+ +	+	+	–
Syncytial knots	+ ++	+ ++	+	+
Syncytial degeneration	+ +	+ +	+	+
Intravillous fibrin deposition	+ ++	+ +	+	+
Perivillous fibrin deposition	+ ++	+ +	+	+
Villous villitis (PBMCs)	+ +	+	+	–
Thrombi	+ +	+ +	+	+
Ectasis	+ +	+ +	+	+
Mural hypertrophy	+ +	+	+	–
Atherothic vessels	+ +	+	+	–
Dystrophic microcalcification	+ +	+ +	+	–

- nil effects seen; + liitle effects; + +moderate effects; + +significant effects; SARS-CoV-2+:Covid positive; SARS-CoV-2-:Covid negative; HIV +ve: HIV positive; HIV-ve: HIV negative

Mineralization of the basement membrane was noted in the HIV positive placenta regardless of SARS-CoV-2 infection (Fig. 1c-d). Syncytial degeneration/necrosis was also evident in all placentae, albeit amplified in the SARS-CoV-2 infected placenta (Fig. 1d-e). Villous microcalcifications (Fig. 1e-f) were noted in the SARS-CoV-2+ HIV+; SARS-CoV-2+ HIV- and SARS-CoV-2- HIV+ groups, being predominant in the COVID+ infected placentae. Villitis and mononuclear inflammatory cells infiltrates (Fig. 2a) was noted in SARS-CoV-2+ HIV+, SARS-CoV-2+HIV- and SARS-CoV-2-HIV+ placentae. Intravillous and perivillous fibrin deposition (Fig. 2b) was diffuse and widespread. Fibrin deposition was both focal and diffuse, occasionally with the avascular core being surrounded by a degenerative, irregular, syncytial trophoblast layer. Distal villous hypoplasia/mesenchymal dysplasia and patchy oedematous villi was also noted (Fig. 2c). Anuclear thinned-out syncytium bulging over stromal capillaries into the intervillous space was also evident. High grade spatial and temporal foetal malperfusion was noted in SARS-CoV-2+HIV+; SARS-CoV-2 +HIV- and SARS-CoV-2- HIV+ placentae. These features included thrombi (Fig. 2d), ectasis (Fig. 2e-f), mural hypertrophy and atherotic vessels.

Fig. 2

Pathological features of SARS-CoV-2+HIV+ infected AAP placental villi showing (a) PBMC infiltration; (b) perivillous and intravillous fibrin; (c) mesenchymal dysplasia; (d) thrombi and (e-f) vascular ectasis (x40).

Discussion

The main findings of this study were increased villitis, syncytiotrophoblast injury, mineralisation of synctiotrophoblast basement membrane, fibrin deposition, microcalcifications, vascular maladaptation and placental weight gain in the SARS-CoV-2+ HIV+ compared to SARS-CoV-2+HIV- and SARS-CoV-2-HIV+ placentae.

Villitis

We report inter- and intra-villous inflammation in placentae with SARS-CoV-2 and HIV infection and in a combination of both. Notably, SARS-CoV-2 infection causes chronic histiocytic intervillitis together with syncytiotrophoblast necrosis (15). The villitis may be an accrual of mononuclear inflammatory cells as previously reported [11]. These monocytoid cells express CD11c, CD14, CD68, and CD163, with some expressing myeloperoxidase+ CD66b+ neutrophils+ , a CD10-immature subgroup [12]. Notably, intervillitis has also been observed in Zika [13], [14] and Dengue viral infections [15]. [[16]]. Villitis within the stroma is indicative of proinflammatory cytokine release, that induces vascular smooth muscle cells apoptosis. Moreover, SARS-CoV-2 related placental inflammation is believed to emanate from elevated IL-6 and TNF-α levels and is associated with endothelial dysfunction, thereby prompting maternal thromboembolic events [17]. We also observed thrombi in all the AAP placentae, being more pronounced in the SARS-CoV-2+ placentae. Occlusive and non-occlusive thrombi have been previously graded by a working group in Amsterdam, highlighting the need for delineating diagnostic placental pathology values [18].

Synctiotrophoblast necrosis

Apart from the maintenance of immune tolerance to fetal cells, syncytiotrophoblasts also serve as an immunological barrier against pathogens. In fact they are reported to sense viral pathogens via toll-like receptors and retinoic acid-inducible gene I-like receptors thereby mediating signal induction of type I interferons [19]. More recently, syncytiotrophoblast stress has been reported to include parvovirus infection albeit minimally, hence it is plausible that both HIV and SARS-CoV-2 infection would also aggravate the stress [20]. Notably, the denuding of the syncytiotrophoblast in our study may permit vertical transplacental foetal transmission of maternal SARS-CoV-2 infection since angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 receptors (TMPRSS2) are expressed on its plasmalemma [21]. Also the syncytiotrophoblast necrosis observed in our study will influence the binding of SARS-CoV-2 to its ACE-2 and TMPRSS2 receptors on its plasmalemma [21].

Mineralisation of Synctiotrophoblast basement membrane

We report pronounced mineralization of the syncytiotrophoblast basement membrane in the SARS-CoV-2+HIV+, SARS-CoV-2+HIV- and SARS-CoV-2-HIV+, indicative of a general viral insult. Sedimentation of minerals, specifically calcium phosphate and haemosiderin on the syncytial basement membrane is related with fibrotic avascular villi [22]. Placental hemosiderin deposition was also previously observed in cytomegalovirus infection [31]. Mineralization of the syncytial basement membrane is associated with foetal vascular malperfusion and correlates with adverse foetal outcomes such as polyhydramnious, placental and fetal hydrops [23].

Fibrin deposition

In our study, extensive fibrin deposition occurred in all placentae, being more prominent in the SARS-Co-V-2+ placentae. Our findings are corroborated by others [16], [24]. This pervasive fibrin deposition may be linked to placental malperfusion and poor fetal outcomes [25], [26], corroborating the viable yet small-for-gestational-age neonates observed in our study. Nonetheless, in alveoli of acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infections there is an accumulation of proteinaceous and fibrin exudate and macrophages [27]. Placental derived mesenchymal stem cells is believed to enhance the immunomodulatory and anti-inflammatory milieu of ARDS [27].

Microcalcifications

We also report an increased prevalence of microcalcifications in the SARS-CoV-2+ AAP placentae as corroborated by others [28]. These calcifications may emanate from syncytial trophoblast damage, or it may reflect a hypercoagulable state caused by SARS-Co-V-2 infection. Apoptotic bodies thereafter undergo osteogenic differentiation as they concentrate calcium and phosphate to allow crystal nucleation and mineralization [29]. Calcification of the placenta increases with gestational age, and may be associated with decreased placental perfusion, infarcts, still births and viral infections [30]. Notably these vascular calcifications represent a multifaceted connection of regulatory pathways such as adenosine signaling, osteochondrogenesis, inflammation, hypoxia, autophagy and phosphate signaling [31], [32].

Vascular pathology

Our findings demonstrate higher levels of vascular malperfusion in the SARS-CoV-2 positive AAP placentae, regardless of HIV status compared to the SARS-CoV-2 negative placentae. COVID-19-infected placentae are associated with aberrant vascular supply [12], [33], and supports malperfusion features of mural hypertrophy, ectasis, atherosis and thrombi observed in our study. Maternal vascular malperfusion anomalies lead to fetoplacental underperfusion as observed in preeclampsia and hypercoagulable states such as antiphospholipid antibodies, lupus anticoagulant, factor V Leiden mutation, protein S and C deficiency [34]. Off note, placental hypoperfusion leads to maternal hypoxia following severe COVID-19 infection. Maternal vascular malperfusion features are more frequent in HIV infected (24.2%) compared to HIV uninfected women (12.6%) [35]. This pathology is associated with the development of hypertensive disorders of pregnancy and is elevated in HIV-infected women receiving highly active antiretroviral therapy (HAART) [36], [37]. Vascular malperfusion is also elevated in women on zidovudine (AZT) as observed in 76.47% out of 51 placentae of HIV positive mothers or nevirapine (NVP) compared to untreated cases [35]. Notably all HIV infected women in our study received anti-retroviral therapy, a standard of care in South Africa.

Placental weight

In our study, the SARS-CoV-2+HIV+ AAP placenta was almost twice the weight of a previously reported intrauterine SARS-CoV-2 infected placenta [38]. Tasca et al., (2021) reported heavier placental weight in COVID positive women receiving antibiotics or antivirals such as chloroquine [38]. It is plausible that SARS-CoV-2/HIV infection in pregnancy directly affects placental growth via viral infection, or indirectly through an exaggerated inflammatory response of the microenvironment. It is also possible that the gross increase in placental size may also be a compensatory proliferation of vascular supply to meet the increased oxygen and nutrient demands of the foetus required to support a viable neonate. Consistent with our findings, placental weight dysregulation [39], [40], villitis, enhanced chorionitis/chorioamnionitis, funisitis membrane inflammation and vasculo-pathology have been reported in HIV infection [41].

Foetal outcomes

Nonetheless, we report good maternal and viable foetal outcomes in the third trimester of both SARS-CoV-2 infected AAPs regardless of their HIV status, indicating that close monitoring and appropriate patient management may avert termination of pregnancy, and prevent adverse maternal and poor foetal outcomes. However, one baby from the SARS-CoV-2+HIV+ AAP in our study was afflicted with dysmorphic facial features and a left foot deformity. This may be a consequence of low amniotic fluid volume and the absence of a “cushion effect”. Notably, AAPs are associated with a high percentage (21%) of birth defects, limb and joint defects, facial and cranial asymmetry and deformity due to foetal compression [42].

Treatment

There is no standardization of treatment principles for AAP and peri-operative treatment options, which would improve newborn survival, and reduce maternal and neonatal morbidity. The diagnosis of AAP therefore creates a conundrum regarding clinical management as most cases are identified after 20 weeks with a live foetus. Decisions made regarding expectant management or immediate laparotomy are taken by both the patient and the attending clinician. Expectant management includes the patient being hospitalized for weeks under close observation because intra-abdominal bleeding cannot be predicted and may occur rapidly. A limitation of our study was the lack of a comparative group of intrauterine pregnancies; these should be investigated in future studies.

Conclusion

This study highlights placental pathology of AAP pregnancies comorbid with SARS-CoV-2 and HIV-1 viral insult. Pathological features include increased mineralisation of the syncytial basement membrane, microcalcifications, and villitis together with evidence of vascular adaptation in the SARS-CoV-2 compared to HIV-1 viral transgression alone. The characteristic of vascular maladaptation such as ectasis and mural hypertrophy indicate a compensatory response to meet the perils of a hypoxic insult of an AAP stemming from a need to meet the oxygen and nutrient demands of the foetus. Notably the pathological features of vascular malperfusion also occur in HIV infected women receiving antiretroviral therapy albeit at a lesser extent. Careful monitoring with suitable patient management will prevent early termination of AAPs co-infected with SARS-CoV-2 and HIV-1, thereby preserving maternal and foetal outcomes.

Funding

This study was funded by productivity funds of Prof T Naicker.

CRediT authorship contribution statement

Dr S Ramphal: Conceptualisation, design, critical revision of scientific content and approval of final version of manuscript. Professor T Naicker: Design, interpretation of data, writing and editing of manuscript, and approval of final version of manuscript. Dr N Govender: Design, interpretation of data, writing, editing and approval of final version of manuscript. Mrs S Singh: Analysis, interpretation of data, editing and approval of final version of manuscript. Dr OP Khaliq: Analysis, editing and approval of final version of manuscript.

Declaration of Competing Interest

All Authors declare that there are no conflicts of interest.