ORIGINAL ARTICLE

Serum Levels of Collectins Are Sustained During Pregnancy:Surfactant Protein D Levels Are Dysregulated Priorto Missed Abortion

Kavita Kale1 & Pallavi Vishwekar2 & Geetha Balsarkar3 & M. J. Jassawalla3 & Saad Alkahtani4 & Uday Kishore5&

Ganpat Sawant2 & Taruna Madan1

Received: 26 December 2019 /Accepted: 6 May 2020# Society for Reproductive Investigation 2020

AbstractAbout 15% of pregnant women undergo missed abortion (MA), wherein women do not experience cramping and vaginalbleeding. Dysregulation of the immunemolecules and steroid hormones contribute to early pregnancy loss. Collectins- surfactantprotein A (SP-A), surfactant protein D (SP-D), and mannose-binding lectin (MBL) are a group of innate immune moleculesregulated by the steroid hormones. Reduced levels of SP-A and SP-D during the early gestation exhibited a significant associationwith the severe early onset preeclampsia. In order to determine the serum profile of collectins throughout the normal pregnancyand explore their predictive potential during the 8–12 weeks of gestation for MA, we examined a prospective cohort of pregnantwomen (n = 221). The serum levels of SP-A and SP-D were significantly downregulated in the normal pregnant women in all thethree trimesters (n = 30) compared with the non-pregnant women (n = 20) and were not significantly different across the threetrimesters. Fourteen of the women from the cohort underwent MA during the 14–20 weeks of gestation and exhibited asignificant downregulation in the serum levels of SP-D during 8–12 weeks of gestation. A significant inhibition of the HTR-8/SVneo cell proliferation and migration in the presence of a recombinant fragment of human SP-D suggested the relevance ofSP-D in placental development.We report here that the serum levels of SP-A, SP-D, andMBL are consistently maintained duringpregnancy in the Indian cohort. Dysregulated serum levels of SP-D and P4/E2 ratio during the early first trimester may predictoccurrence of MA.

Keywords Collectins . Steroid hormones . Pregnancy .Missed abortion . Surfactant protein D

Introduction

Regulation of the maternal immune response to semi-allogenic fetus is the key to a successful pregnancy. Both thelocal and systemic immune responses are active during thepregnancy that are controlled by the steroid hormones in orderto facilitate the fetal growth [1]. Dysregulation of this intricatebalance of the immune response and hormones may lead topregnancy-related complications. Spontaneous abortion is themost common complication in the first trimester and its inci-dence in India varies between 10 and 32% [2]. Altered firsttrimester serum/plasma levels of several endocrine (hCG, pro-gesterone, estradiol) and immune (TNF-α, IFN, IL-10, IL-6)markers have the potential for prediction of threatened abor-tion [3, 4]. However, the studies on the prediction of missedabortion (MA) are limited. About 15% of pregnant womenundergo a missed abortion, wherein women do not experience

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s43032-020-00209-3) contains supplementarymaterial, which is available to authorized users.

* Taruna Madantaruna_m@hotmail.com

1 Department of Innate Immunity, ICMR-National Institute forResearch in Reproductive Health, J.M. Street, Parel,Mumbai, Maharashtra 400012, India

2 Department of Obstetrics and Gynaecology, DY Patil MedicalCollege, Hospital and Research Centre, Nerul, NaviMumbai, Maharashtra, India

3 Department of Obstetrics and Gynaecology, Nowrosjee WadiaMaternity Hospital, Parel, Mumbai, Maharashtra, India

4 Department of Zoology, College of Science, King Saud University,Riyadh, Saudi Arabia

5 Biosciences, College of Health and Life Sciences, Brunel UniversityLondon, Uxbridge, UK

Reproductive Scienceshttps://doi.org/10.1007/s43032-020-00209-3

the typical miscarriage symptoms such as cramping and vag-inal bleeding as reported in the case of a threatened abortion[5]. Decreased first trimester progesterone, macrophage inhib-itory cytokine-1 (MIC-1) and pregnancy-associated plasmaprotein-A (PAPP-A) have been shown to predict the occur-rence of MA [6, 7]. Approximately 50% of miscarriages areassociated with the chromosomal abnormalities, whereas theremaining are associated with the implantation failure andseverely impaired development of the feto-maternal interface[8]. Identifying a set of molecular markers predicting MAduring early stages of pregnancy may enable the use of ther-apeutic intervention such as progesterone [9] and appropriatecounseling of the pregnant women. As per ACOG guidelinesand a recent Cochrane review, there is no strong evidence forthe usefulness of any of the preventative therapeutic interven-tions [10, 11]. Identification of the predictive markers for MAcan facilitate the identification of new potential therapeutictargets for preventive strategies.

Collectins such as surfactant protein A (SP-A), surfactantprotein D (SP-D), and mannose-binding lectin (MBL) ex-press differentially at the feto-maternal interface of womenundergoing spontaneous abortion and in the abortion pronemurine model [12]. Collectins (Ca2+-dependent C-typelectins), a class of soluble pattern recognition proteins secret-ed by the mucosal epithelial cells, are integral to the mainte-nance of immune homeostasis and host defense [13, 14]. Thevillous, extravillous placental trophoblast, and decidual stro-mal cells express SP-A, SP-D, and MBL [15, 16]. We haveshown an increased expression of SP-D at the feto-maternalinterface of women undergoing spontaneous labor [16].Recently, we reported an increased expression of collectinsin the term human placenta of women with severe early onsetpreeclampsia (EOPE) [17]. Delayed parturition and a de-creased expression of inflammatory and contractile geneshave been observed in the SP-A and SP-D gene-deficientmice [18]. In vitro treatment of the murine decidual cells withrecombinant human SP-A and SP-D reduced the number ofLPS-induced inflammatory macrophages [19]. These evi-dences implicate collectins in the regulation of immune re-sponse at the feto-maternal interface. Importantly, Candaet al. reported reduced MBL immunoreactivity in the tropho-blast and decidual tissues of first time pathologic first trimes-ter miscarriage patients [20]. The MBL2 gene polymor-phisms leading to low serum MBL levels in Brazilian womenwith rheumatoid arthritis are a significant risk factor for mis-carriage [21]. A significant decrease in the serum SP-A, SP-D, and P4/E2 ratio at 10–20 weeks of gestation in the womenwith subsequent development of severe EOPE implicated animportant role of SP-A and SP-D in the placental develop-ment [17]. Since the pathophysiological mechanisms of mis-carriage and preeclampsia are relatively similar with a differ-ent degree of placental damage [8], it prompted us to deter-mine if the serum levels of collectins are dysregulated in the

first trimester of asymptomatic women destined to undergospontaneous abortion (MA).

With respect to the systemic levels of collectins duringpregnancy, a significantly increased maternal serum MBLand activation of the lectin pathway has been reported duringthe early stages of normal pregnancy [22]. Thematernal serumSP-A levels significantly increased after labor [23]. SP-A, SP-D, and MBL proteins were detected in human amniotic fluid(AF) at 26 weeks of gestation with a significant increase after32 weeks of gestation [24, 25]. However, there are no reportson the serum levels of SP-A and SP-D across different phasesof pregnancy.

It is well documented that the expression of collectins in thefemale reproductive tract is regulated by the ovarian steroidhormones [26–28]. Importantly, SP-D−/− mice exhibit alteredserum levels of the ovarian hormones with a prolonged estruscycle and increased pre-implantation loss [29]. Serum levelsof progesterone and estradiol progressively increase duringthe pregnancy and are known to orchestrate the immune mod-ulations critical for a successful pregnancy outcome [30, 31].Serum levels of SP-A and SP-D were shown to be positivelyregulated with the P4/E2 ratio in preeclampsia [17]. Thus, itwas pertinent to evaluate the serum profile of collectins andtheir correlation with the steroid hormones during normalpregnancy and in MA.

In the present study, serum samples were prospectivelycollected from a cohort of 221 Indian pregnant women inorder to investigate the profile of collectins during pregnancy,before an impending MA and their correlation with the levelsof steroid hormones. We report here that the maternal serumlevels of collectins were consistently maintained across thethree trimesters, and SP-D levels were significantly downreg-ulated in the asymptomatic pregnant women with subsequentspontaneous abortion (MA). Furthermore, with an aim to un-derstand the role of SP-D in placental development, we eval-uated the migration and proliferation of HTR-8/SVneo cells(first trimester trophoblastic cell line) treated with a recombi-nant fragment of human SP-D (rfhSP-D).

Materials and Methods

Ethics Statement

Serum samples were obtained with written informed consentof the participants, and the protocol was approved by theInstitutional Ethical Committee of Indian Council ofMedical Research-National Institute for Research inReproductive Health (ICMR-NIRRH), Mumbai (No. 276/2015), Ethics Committee for Research on Human Subjectsof DY Patil Hospital, Nerul, Navi Mumbai (PDYPMC/Ethics/2015), and Wadia Maternity Hospital, Parel, Mumbai(IEC-NWMH/AP/2015/003-version 2).

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Study Population

Based on the incidence rate of spontaneous abortions in India(10–32%) [2], a prospective study was designed using a co-hort of healthy Indian pregnant women (n = 221). It was con-sidered that at least 22 of these women participants may un-dergo spontaneous abortion of which some may show nosymptoms (MA). The study participants were recruited fromMay 2015 to February 2016 at the Department of Obstetricsand Gynaecology of DY Patil Hospital, Navi, Mumbai, andWadia Maternity Hospital, Mumbai, during their first antena-tal care (ANC) visit. Blood samples (5 ml) were collectedfrom these participants progressively during their regularANC visits over all the three trimesters (trim) of pregnancy(1st trim, 8–12: 2nd trim, 20–24; 3rd trim, 28–32 weeks ofgestation) and at post-partum (within 2 weeks). Out of the 221study participants, the normal pregnant women (P) withhealthy, uncomplicated pregnancies, who provided samplesin all the three trimesters and at post-partum (PP) along withcomplete documentation (n = 30) were included as group I.Fourteen asymptomatic women, who provided samples atthe early stages of pregnancy (8–12 weeks of gestation) andsubsequently underwent spontaneous abortion (MA) (be-tween 14th and 20th week of gestation), formed the groupII. The diagnosis of abortion was based on the ultrasoundfindings and was defined as spontaneous loss of pregnancy(i.e., absent fetal cardiac activity) at less than 20 weeks ofgestation [10]. The detailed study design is provided inFig. 1. The number of participants in groups I and II werelimited owing to the loss during follow-up (n = 75), withdraw-al of participants from the study (n = 31), and exclusion of

participants owing to the following reasons (n = 56). Womenparticipants who could not provide all the four blood samplesand with incomplete documentation were excluded from thefinal analysis. On clinical examination, the women whoshowed any infections during pregnancy, autoimmune dis-ease, thyroid-related disorders, pregnancy-related complica-tions (e.g., threatened abortion, gestational diabetes, chronichypertension, gestational hypertension, and preeclampsia)other than asymptomatic spontaneous abortion (MA) wereexcluded from the study. Study participants treated with hor-mones or any other medications were also excluded.

It is to be noted that the hospitals involved in thepresent study were tertiary referral hospitals, and hence,many participants preferred to go to a local hospital fortheir subsequent visits. Furthermore, owing to an Indiantradition, the pregnant women opt for a hospital neartheir mother’s place for delivery in order to get betterpost-partum care. These inevitable reasons contributedto the loss to follow-up observed in our study.

Blood samples were collected during the mid-luteal phasefrom the age-matched, healthy, regularly cycling, non-pregnant (NP) study participants (group III, n = 20), recruitedfrom the women visiting the clinic for contraception guidance.

Serum Samples

Peripheral blood from participants was collected in a serumseparator tube, and the samples were allowed to clot for30 min before centrifugation at 1000×g for 15 min. Serumwas separated, aliquoted, and stored at − 80 °C until analysis.Repeated freeze-thaw of the serum samples was avoided.

Fig. 1 The Prospective studydesign to evaluate the maternalserum levels of collectins andsteroid hormones duringpregnancy and at early firsttrimester in missed abortion (MA)

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Measurement of Serum Levels of Collectins (SP-A, SP-D and MBL) and Steroid Hormones (17β-Estradiol,Progesterone and Free Testosterone)

Serum levels of SP-A, SP-D and MBL were measured usingthe commercially available (sandwich) enzyme linked immu-nosorbent assay (ELISA) kits (Human SP-D Duoset ELISAkit and HumanMBLELISA kit, R&DSystems, USA; HumanSP-A ELISA kit, Bio Vendor, Czech Republic) according tothe manufacturer’s instructions.

Serum levels of the steroid hormones (17β-estradiol (E2),progesterone (P4), and free testosterone (FT)) were measuredby the competitive ELISA kits using the manufacturer’s pro-tocol (Human 17β-estradiol ELISA kit, Human progesteroneELISA kit, Human free testosterone ELISA kit, DiagnosticBiochem Canada Inc., Canada). The estradiol assay kit had afunctional sensitivity of 10 pg/ml with an assay range of 20–3200 pg/ml. The progesterone assay kit had a functional sen-sitivity of 0.1 ng/ml with an assay range of 0.3–60 ng/ml. Thefree testosterone assay kit had a functional sensitivity of0.17 pg/ml with an assay range of 0.25–125 pg/ml.

Both intra-assay and inter-assay coefficients of variationwere < 10% for all the six kits used for the study. The absor-bance was measured using an ELISA reader at 450 nm(mQuant, Biotek Instruments Inc., Germany).

Cell Culture

HTR-8/SVneo cell line (kindly provided by Dr. Satish KGupta, National Institute of Immunology, New Delhi, India)was maintained in Dulbecco’s modified Eagle medium(DMEM) and Ham’s Nutrient Mixture F12 medium(HAM’s F12, Sigma-Aldrich, USA) in 1:1 ratio supplementedwith 10% heat inactivated fetal bovine serum (FBS, Gibco®,USA) and antibiotic-antimycotic cocktail–containing penicil-lin (100 units/ml), streptomycin (100 μg/ml), andamphotericin B (0.25 μg/ml) (Gibco®, USA) at 37 °C in ahumidified chamber containing 5% CO2. The HTR-8/SVneocell line was established from extravillous trophoblast cellsderived from the human first trimester placenta and immortal-ized by SV40 large T antigen [32]. Cells were treated withGeneticin (G418 Sulpfate) (Gibco®, USA) at 100 μg/ml afterevery 3rd passage to avoid the growth of untransfected HTR-8/SVneo cells with SV40 large T antigen [32].

Immunofluorescence

HTR-8/SVneo cells (1.5 × 104) were grown on the coverslipsand air-dried followed by fixation with methanol (Qualigens,India) for 10 min at − 20 °C and permeabilization using 0.1%v/v Triton X-100 (Sigma, USA) for 15 min at room tempera-ture. Cells were then blocked with 3% w/v bovine serumalbumin (BSA) (GeNei TM, India) for 30 min and incubated

with the rabbit polyclonal antibody against human SP-D, SP-A,andMBL in phosphate-buffered saline (PBS) (1:25, Santa CruzBiotechnology, USA) and cytokeratin 7 in PBS (1: 250,Abcam, USA) overnight at 4 °C. Negative control sectionswere incubated with PBS alone instead of the primary anti-body. Cells were further incubated with the goat polyclonalanti-rabbit IgG-FITC-tagged secondary antibody (1:200,Invitrogen, USA) for 40 min at room temperature. After finalrinse in PBS, 4′,6-diamidino-2-phenylindole (DAPI) was usedto stain the cell nuclei and mounted in vectashield (VectorLaboratories, Inc., USA). Images were captured on the fluores-cence microscope (Carl Zeiss, Germany) at × 20magnification.

rfhSP-D Preparation

The recombinant fragment of human SP-D (rfhSP-D) used inthis study was expressed in Escherichia coli, purified andcharacterized as described previously [33]. The rfhSP-D com-prises of a functional homotrimeric C-type lectin domainfollowed by the neck region and eight collagen Gly-X-Y re-peats. The endotoxin levels in the rfhSP-D preparation weredetermined using the QCL-1000 Limulus amebocyte lysatesystem (BioWhittaker Inc., USA). The amount of endotoxinpresent in the preparations was estimated to be < 4 pg/μg ofrfhSP-D.

Trophoblast Wound Healing Assay

In order to understand the functional relevance of levels ofcollectins during early pregnancy, we performed in vitro as-says with HTR-8/SVneo cells (first trimester trophoblastic cellline), a model for the study of placental function. SP-D hasbeen reported as a potent anti-tumor molecule against varioustumor cell lines [33]. In view of the similarity of the pathwaysinvolved in tumor progression and placental development[34], the effect of SP-D on trophoblast cell proliferation andmigration was evaluated using cell proliferation and cell mi-gration assay.

Wound healing assay was performed to assess the effect ofrfhSP-D treatment on the HTR-8/SVneo cell migration. Weused rfhSP-D at the concentration of 10 μg/ml, previouslyreported to be inducing apoptosis and cell death in cancer cells[34]. It is important to note that the concentration of rfhSP-Dused in this experiment is 3-log fold higher than the humanserum concentration of free SP-D. SP-D has several ligands inserum including saccharides, albumin, and globulins that limitthe amount of free SP-D in serum [35, 36]. HTR-8/SVneocells (2.5 × 105) per well were seeded in the 12-well tissueculture plate in 1 ml of DMEM/F-12 phenol red–free mediumsupplemented with 10% FBS and incubated at 37 °C in 5%CO2 until 100% confluence. After 24 h of serum starvation,the cells were treated with mitomycin C (10 μg/ml) [37] at37 °C for 2 h to suppress the cell proliferation and scratched

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using a sterile tip. Cells were washed twice with the incom-plete medium and treated with rfhSP-D (10 μg/ml) inDMEM/F-12 phenol red–free medium supplemented with2% charcoal-stripped serum for 16 h. The images ofpreselected fields were captured at 0 and 16 h time pointsusing an inverted microscope (Nikon Corporation, Japan).Wound size was measured using the NIS Elements, BR 3.0software, and the percent wound closure was calculated usingformula a − b/a × 100, where a is the distance between edgesof the wound at the 0 time point, and b is the distance at 16-htime point [38]. The experiment was performed three timeswith a duplicate in each experiment.

Cell Viability Assay

MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazoliumbromide; Calbiochem) assay was used to investigate the cellviability. HTR-8/SVneo cells (1 x 104) per well were plated in96-well plates and incubated with the rfhSP-D (10 μg/ml).After treatment, 10 μl ie ul of 5 mg/mlMTT solution preparedin the DMEM/F-12 incomplete medium was added to eachwell. After incubation for 2 h at 37 °C, the formazan crystalswere dissolved in acidified 2-propanol (100 μl/well) and theabsorbance was measured at 570 nm (mQuant, BiotekInstruments Inc., Germany).

Statistics

All the statistical analyses were performed using GraphPadPrism version 5.0 (GraphPad Software, San-Diego, CA, US)and MS Excel. Gaussian distribution of data was confirmedusing the Kolmogorov-Smirnov test and Shapiro-Wilk normalitytest. For data with non-normal distribution and small sample size,non-parametric Mann-Whitney test was used to analyze the dif-ferences between two groups, and Kruskal-Wallis test withDunn’s post hoc test was used to compare the differences be-tween more than two groups. One way ANOVA with Tukey’spost hoc test was used to analyze the differences between morethan two groups when the data was normally distributed.Correlation analysis between the collectins and P4/E2 ratio wasevaluated using the non-parametric Spearman’s rank correlationcoefficient. Receiver operating characteristic (ROC) curves weredrawn for each variable (SP-D and P4/E2) to elicit the optimumsensitivity, specificity, and cut-off values. The Fisher’s exact testwas used to analyze the categorical variables (e.g., gravida) be-tween MA and controls. ELISA data were analyzed andexpressed as median (interquartile range (IQR)). The demo-graphic data was reported as mean ± SEM (standard error ofthe mean) and values of p < 0.05 were considered statisticallysignificant. Wound healing, viability, and real-time assay dataof HTR-8/SVneo cells were analyzed using one-way ANOVAwith Tukey’s post hoc test and expressed as mean ± SEM.

Results

Characteristics of the Study Population

Out of the 221 recruited participants, 30 normal pregnantwomen provided blood samples in all the three trimestersand post-partum (group I). A total of 14 asymptomatic partic-ipants that underwent spontaneous abortion (MA) in the 2ndtrimester (group II) had provided blood samples in the firsttrimester (8–12 weeks) (Fig. 1). Women in the two groupswere not significantly different with respect to the demograph-ic and clinical characteristics (Table 1). The mean gestationalage of the group I participants at the time of delivery was 37.9± 0.19 weeks. A total of 10 cesarean sections and 20 normaldeliveries were performed. All the 30 neonates (from group Iparticipants) were born healthy without asphyxia. A total of20 non-pregnant study participants formed the group III.Serum samples of participants from groups I, II and III wereused for ELISA analysis.

Significantly Decreased Serum Levels of SP-A and SP-D Are Maintained During the Pregnancy

Figure 2a shows the progressive serum levels of collectinsduring the course of normal pregnancy. The serum levels ofSP-A were consistently decreased in all the three trimesters ofpregnancy when compared with the non-pregnant women(p < 0.001). SP-A levels post-partum were not significantlydifferent from the non-pregnant women (p > 0.05). The serumSP-A levels post-partumwas significantly higher than its levelin the three trimesters of pregnancy (p < 0.001) (Fig. 2a-i,Table 2). Serum levels of SP-D were consistently decreasedin all the three trimesters of pregnancy (p < 0.001) comparedwith the non-pregnant women. Serum SP-D levels post-partum were not significantly different compared with theirlevels in the 1st and 2nd trimesters (p > 0.05), whereas differsignificantly compared with their levels in the 3rd trimester(p < 0.05) (Fig. 2a-ii, Table 2). Serum MBL levels in all thethree trimesters of pregnancy were not significantly differentwhen compared with the non-pregnant women (p > 0.05).However, serum MBL levels decreased significantly post-partum compared with the non-pregnant women (p < 0.05).MBL levels in the 1st and 3rd trimester were not significantlydifferent when compared with the levels post-partum(p > 0.05), whereas in the 2nd trimester, levels were increasedsignificantly compared with the post-partum (p < 0.01) (Fig.2a-iii, Table 2). Levels of none of the three collectins weresignificantly different among the three trimesters (p > 0.05)and were consistently maintained (Fig. 2a-i, ii, iii).

As serum levels of the collectins were maintained during thethree trimesters, we further analyzed the mean serum levels ofSP-A, SP-D and MBL in pregnancy compared with their levelspost-partum andwith the non-pregnantwomen. The serum levels

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of SP-A were significantly decreased in the normal pregnancythan the non-pregnant women (p< 0.001) and the post-partumlevels (p< 0.001) (Fig. 2b-i, Table 2). Serum levels of SP-Dwerealso significantly decreased in the normal pregnancy comparedwith the non-pregnant women (p< 0.001). Although the serumSP-D level showed a moderate increase post-partum, it did notdiffer significantly when compared with normal pregnancy(p> 0.05) (Fig. 2b-ii, Table 2). The serum MBL levels of thepregnant women were not significantly different from the non-pregnant women (p > 0.05). However, they were increased sig-nificantly compared with the levels post-partum (p< 0.05) (Fig.2b-iii, Table 2).

SP-D Levels Significantly Decreased at Least 2 WeeksPrior to an Impending Missed Abortion

We retrospectively analyzed the levels of collectins in the 1sttrimester (8–12 weeks) serum samples of asymptomatic wom-en (group II, n = 14) with subsequent spontaneous abortions(MA) during 14–20 weeks of gestation. Levels of SP-D weresignificantly reduced in the “MA”women than the gestationalage-matched normal pregnant women (n = 30) during earlypregnancy (p < 0.01). However, differences in the levels ofSP-A and MBL (p > 0.05) were not significant between thetwo groups (Fig. 3a, b, and c, Table 2). Further, the ROC

Fig. 2 Serum levels of the collectins (i) SP-A, (ii) SP-D, and (iii) MBLduring different stages of pregnancy (group I, n = 30) and in non-pregnantwomen (group II, n = 20) (a); during pregnancy (n = 90, 30 samples ofeach trimester), post-partum (n = 30), and in non-pregnant women (n =20) (b). SP-A, surfactant protein A; SP-D, surfactant protein D; MBL,

mannose-binding lectin; PP, post-partum (within 2 weeks); NP, non-pregnant women. 1st trimester, 8–12 weeks; 2nd trimester, 20–24 weeks; 3rd trimester, 28–32 weeks. Data represented as median(interquartile range). *p < 0.05; **p < 0.01; ***p < 0.001; ns, notsignificant using Kruskal-Wallis test with Dunn’s post hoc test

Table 1 Demographic and clinical characteristics of the study groups

Group I Group II Group III p valueNormal pregnant women (P) Missed abortion (MA) Non-pregnant women (NP)(n = 30) (n = 14) (n = 20)

Mean age (years) (SEM) 24.8 (0.70) 23.1 (0.59) 27.6 (0.59) *#p > 0.05GravidaPrimigravida (%) 26 (86.6) 10 (71.4)Multigravida (%) 4 (13.3) 4 (28.6) - #p > 0.05

Mean gestational age at the time of delivery (weeks) (SEM) 37.9 (0.19) - - -Mode of deliveryC-section (%) 10 (33.3) - - -Normal vaginal delivery (%) 20 (66.6)

Mean birth weight (grams) (SEM) 2971 (63.44) - - -Child sex (%)Male 16 (53.3) - - -Female 14 (46.6)

* Represents p value between normal pregnant women and non-pregnant women# Represents p value between normal pregnant women and missed abortion

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curve of SP-D levels at 8–12 weeks of gestation showed anoptimum cut-off at 725.5 pg/ml (sensitivity of 57.14% andspecificity of 93.3%) with a significant area under the curve(p < 0.01) in women with subsequent MA during 14–20 weeks of gestation (Fig. 4a).

Progressive Increase in the Levels of SteroidHormones During Normal Pregnancy

Serum levels of E2, P4, and FT in the healthy pregnant women(n = 30) were measured during all the three trimesters of preg-nancy and post-partum. Figure 5 shows progressively in-creased serum levels of the steroid hormones during thecourse of normal pregnancy. Serum levels of E2 increasedsignificantly during the 1st, 2nd, and 3rd trimesters of preg-nancy compared with the levels post-partum (p < 0.05,p < 0.001). (Fig. 5a-i, Table 3). The serum levels of P4 werefound to be increased significantly in all the three trimesters ofpregnancy compared with the levels post-partum (p < 0.01,p < 0.001) (Fig. 5a-ii, Table 3). The serum levels of FT werealso increased significantly in the 3rd trimester of pregnancy

(p < 0.001) with no significant change in the 1st and 2nd tri-mester (p > 0.05) compared with the levels post-partum(Supplementary Fig. S1A-i, Supplementary Table S1).

Decreased Levels of 17β-Estradiol, Progesterone,P4/E2 Ratio, and Increased Free Testosterone at Least2 Weeks Prior to an Impending Missed Abortion

The serum levels of E2 and P4 showed a significant decline inwomen with “MA” (n = 14) during early pregnancy comparedwith the gestational age-matched normal pregnant women(n = 30) (p < 0.001) (Fig. 5b-i, 5b-ii, Table 3). Levels of theFT were found to be increased significantly in women with“MA” compared with the normal pregnant women (p < 0.001)(Supplementary Fig. S1B-i, Supplementary Table S1).

Consistently Maintained P4/E2 Ratio During NormalPregnancy

Ratio of the serum levels of P4 to E2 was determined in thepregnancy and post-partum. Although P4 and E2 levels

Table 2 Serum levels of collectins in pregnancy and missed abortion

Sr no. Parameters Group I Group II Group III p value

Pregnancy (P)(n = 90), n = 30,each trimester

Post-partum(PP) (n = 30)

Non-pregnant(NP) (n = 20)

Missedabortion (MA)(n = 14)

1 SP-A (ng/ml) 17.7 (12.1–21.6) 24.9 (21.3–31.3) 24.4 (21.2–30.5) - *p < 0.001, #p < 0.001, $p > 0.05

1st trimester 17.7 (12.3–22.1) - - 14.6 (13.2–20.9) ap < 0.001, bp < 0.001, cp > 0.05

2nd trimester 17.4 (11.9–20.5) - - - ap < 0.001, bp < 0.001

3rd trimester 18.1 (11.9–22.2) - - - ap < 0.01, bp < 0.001

2 SP-D (pg/ml) 1384 (1036–2272) 2178 (1094–3201) 4271 (2966–5069) - *p < 0.001, #p > 0.05, $p < 0.01

1st trimester 1405 (1202–2298) - - 706 (470–1372) ap < 0.001, bp > 0.05 cp < 0.01

2nd trimester 1595 (1213–3184) - - - ap < 0.01, bp > 0.05

3rd trimester 1189 (757–1546) - - - ap < 0.001, bp < 0.05

3 MBL (ng/ml) 550 (391–886) 423 (174–578) 809 (398–1639) - *p > 0.05, #p < 0.01, $p < 0.01

1st trimester 509 (378–1116) - - 986 (436–1235) ap > 0.05, bp > 0.05, cp > 0.05

2nd trimester 735 (451–1520) - - - ap > 0.05, bp < 0.01

3rd trimester 541 (367–745) - - - ap > 0.05, bp > 0.05

SP-A, surfactant protein A; SP-D, surfactant protein D; MBL, mannose-binding lectin

1st trimester (8–12 weeks of gestation), 2nd trimester (20–24 weeks of gestation), 3rd trimester (28–32 weeks of gestation)

*Represents p value between pregnancy and non-pregnant women# Represents p value between pregnancy and post-partum$Represents p value between post-partum and non-pregnanta Represents p value between different trimesters and non-pregnantb Represents p value between different trimesters and post-partumcRepresents p value between missed abortion and 1st trimester

Italic fonts indicate significant variables

Data are shown as the median (interquartile range)

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increased progressively during the pregnancy, the P4/E2 ratioremained constant in all the three trimesters with no signifi-cant change among the trimesters (1st, 18.4 (13.5–30.1); 2nd,21.3 (12.2–36.2); 3rd, 29.2 (17.3–37.7), p > 0.05). The P4/E2ratio declined significantly post-partum compared with theP4/E2 ratio in all the three trimesters of pregnancy (PP, 2.34(0.93–5.28); p < 0.001) (Fig. 5a-iii). Importantly, the P4/E2ratio of 22.95 (14.0–34.6) in the pregnancy and 2.34 (0.93–5.28) at the post-partum were comparatively lower than thepreviously reported mean P4/E2 ratio in the luteal phase of thenon-pregnant women of Indian origin (~ 106.0) and of otherethnicity (Nigeria (~ 85.71) and America (~ 138.4)) [39–41].Unlike consistently maintained serum P4/E2 ratio throughoutthe pregnancy, P4/FT and E2/FT ratio showed progressiveincrease during the pregnancy with a significant declinepost-partum (p < 0.05, p < 0.001) (Supplementary Fig. S1A-ii, iii; Supplementary Table S1).

P4/E2 Ratio Significantly Decreased at Least 2 WeeksPrior to an Impending Missed Abortion

Analysis of the P4/E2 ratio at 8–12 weeks in the women with“MA” showed significant decline compared with the normalpregnant women (MA, 11.0 (0.26–22.2) vs P, 18.4 (13.5–30.1); p < 0.01) (Fig. 5b-iii). Further, ROC curve analysis to

evaluate the screening performance of serum levels of P4/E2ratio for the later occurrence of “MA” at 8–12 weeks of ges-tation showed significant area under the curve (p < 0.001).The best cut-off value for the serum levels of P4/E2 ratio at8–12 weeks of gestation was 9.6 with a sensitivity of 50% andspecificity of 100% in Indian cohort (Fig. 4b). Both P4/FT andE2/FT ratio were also significantly decreased in women with“MA” compared with the normal pregnant women (p < 0.001)(Supplementary Fig. S1B-ii, iii; Supplementary Table S1).

P4/E2 Ratio During Early Pregnancy Is SignificantlyCorrelated with the Serum Levels of SP-D and MBL

As expected, the consistently maintained levels of SP-A, SP-D and MBL did not show any correlation with increasingsteroid hormone levels during the three trimesters of pregnan-cy (data not shown). Hence, we analyzed the correlation be-tween serum levels of collectins and the progesterone to estra-diol ratio (P4/E2) during the pregnancy alone as well as duringthe pregnancy and post-partum together. No significant corre-lation was observed between serum collectin levels and P4/E2ratio during normal pregnancy (data not shown). The P4/E2ratio during pregnancy and post-partum showed no significantcorrelation with SP-D and MBL (data not shown), whereassignificant negative correlation was observed with the serum

Fig. 4 Receiver operating characteristic (ROC) curve for evaluating thescreening performances of the serum levels of SP-D and P4/E2 ratio at 8–12 weeks of gestation for predicting “MA.” The area under the ROCcurve (AUC) and the 95% confidence interval (CI) of SP-D (a) and P4/E2 ratio (b) at 8–12 weeks for predicting “MA” was significantly larger.

The arrow indicates the best cut-off level of (a) SP-D, which yielded asensitivity of 57.14% and specificity of 93.3%; P4/E2 ratio (b) with asensitivity of 50% and specificity of 100% for the impending “MA.”MA,missed abortion; P4: progesterone; E2, estradiol; SP-D, surfactant proteinD; wks, weeks

Fig. 3 Serum levels of the collectins, (a) SP-A, (b) SP-D, and (c) MBLduring 8–12 weeks of gestation in normal pregnant women, P (group I,n = 30) and in womenwho underwent missed abortions, “MA” (group III,n = 14) later during 14–20weeks of gestation. SP-A, surfactant protein A;

SP-D, surfactant protein D; MBL, mannose-binding lectin. Datarepresented as median (interquartile range). **p < 0.01; ns, notsignificant using Mann-Whitney test

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levels of SP-A (r = − 0.33; p = 0.0002) (Fig. 6a). Similar to P4/E2, both P4/FT and E2/FT ratio showed significant negativecorrelation with serum SP-A levels during pregnancy andpost-partum (Supplementary Table S2).

We then evaluated the correlation of levels of P4, E2, FT,P4/E2, P4/FT, and E2/FT ratio with the collectin levels duringthe early stages of pregnancy (8–12 weeks) in the normalpregnant and MA women together. Both E2 and P4 werepositively correlated with SP-D at 8–12 weeks in normal preg-nancy and “MA,” whereas FT was negatively correlated(Supplementary Table S2). The P4/E2 ratio showed a signif-icant positive correlation with the serum levels of SP-D (r =0.32; p = 0.033) (Fig. 6b) and a negative correlation withMBL (r = − 0.45; p = 0.0022). No significant correlation wasobserved with the serum levels of SP-A (r = 0.25; p = 0.11)(data not shown). Significant positive correlation was seen forP4/FT and E2/FT ratio with SP-D at 8–12 weeks in normalpregnancy and “MA” (Supplementary Table S2).

Collectins Are Localized in the Plasma Membrane andCytoplasm of HTR-8/SVneo Cells

Expression of collectins has been previously documented forvillous, extravillous cytotrophoblasts, and trophoblastic cell col-umns [12, 16]. However their expression in trophoblast-derivedcell lines was not known. HTR-8/SVneo cells showed positivestaining for SP-A, SP-D, and MBL proteins (Fig. 7a-v, vi;Supplementary Fig. S2). All three collectins were present in theplasma membrane and cytoplasm, as revealed by immunocyto-chemistry. Cytokeratin 7 (CK7) was used as an epithelial(trophoblastic) cell marker (Fig. 7a-ii, iii).

Recombinant fragment of human SP-D (rfhSP-D)Inhibited the Trophoblast Cell Proliferation andMigration

The effect of rfhSP-D on trophoblast cell migration was eval-uated by wound healing assay. MTT assay was used to eval-uate the effect on trophoblast cell proliferation. Following16 h treatment with rfhSP-D (10 μg/ml), which was three-log higher concentration than serum concentration of freeSP-D, it did not affect cell proliferation whereas 24 h treat-ment led to a significant reduction (28.5%, p < 0.05) in the cellproliferation (Fig. 7d). Trophoblast cell migration evaluated at16 h was reduced significantly (40.6%, p < 0.001) with rfhSP-D (10 μg/ml) treatment (Fig. 7b, 7c). Mitomycin C treatment(10 μg/ml) was used to suppress the cell proliferation, so as toconfirm that the impact observed on wound healing is specif-ically due to cell migration.

Discussion

With a significant alteration in the maternal serum levels ofSP-D and P4/E2 ratio at least 2 weeks before the occurrence ofMA, our study implicates potential of measuring serum levelsof SP-D along with P4/E2 ratio to predict “MA” in Indianpregnant women. We propose that SP-D, along with P4/E2ratio may synergize with the predictive performance of otherpotential biomarkers (progesterone, PAPP-A, and MIC-1) [6,7] for missed abortions. Decreased trophoblast cell migrationand proliferation in the presence of rfhSP-D supports the im-portance of SP-D for placental development. A significant

Fig. 5 Serum levels of the steroid hormones, (i) 17β-estradiol (E2), (ii)progesterone (P4), and (iii) P4/E2 ratio during different stages ofpregnancy and post-partum (group I, n = 30) (a); during 8–12 weeks ofgestation in normal pregnant women P, (group I, n = 30) and in womenwho underwent missed abortion “MA” (group III, n = 14) (b). 1st

trimester, 8–12 weeks; 2nd trimester, 20–24 weeks, 3rd trimester, 28–32 weeks; PP, post-partum (within 2 weeks). Data represented as median(interquartile range). *p < 0.05, **p < 0.01, ***p < 0.001, ns, non-significant using Kruskal-Wallis test with Dunn’s post hoc test

Reprod. Sci.

sustained decrease in the serum levels of SP-A and SP-D butnot of MBL, during normal pregnancy suggests that pregnan-cy has a specific and differential effect on the systemic levelsof collectins. To the best of our knowledge, this is the firstreport that documents systemic levels of immunoregulatorycollectins across the three trimesters and their correlation withP4/E2 ratio during the normal pregnancy and “MA.”

Pregnancy influences the systemic levels of immunoregu-latory proteins such as C1q, galectins, and MBL. Importantly,akin to SP-A and SP-D, serum levels of these proteins alsoremained sustained throughout the pregnancy with no signif-icant change among the trimesters [22, 42–44]. Similar to thatof SP-D, significantly decreased maternal serum levels of

galectin 1, macrophage inhibitory cytokine (MIC-1) andpregnancy-associated plasma protein A (PAPP-A) have beenreported in the first trimester serum sample of women withsubsequent “MA” [6, 7, 45]. A fine balance between immunetolerance and activation is essential for the success of preg-nancy. It has been reported that during pregnancy, adaptiveimmune response is modulated and sustained across the threetrimesters to induce tolerance [46]. We hypothesized thatsustained levels of innate immune molecules such ascollectins could be necessary to maintain immune toleranceas well as to keep the infections in check. Downregulation ofSP-D during early pregnancy in “MA” women in the presentstudy and previously in women with severe EOPE [17]

Table 3 Serum levels of E2, P4, and P4/E2 ratio in pregnancy and missed abortion

Sr no. Parameters Group I Group II p valuePregnancy (P) (n = 30) Post-partum

(PP) (n = 30)Missed abortion(MA) (n = 14)

1 17β-Estradiol, E2 (pg/ml) - 638 (467–952) - *p < 0.05, #p < 0.001, $p < 0.001

1st trimester 1340 (1162–1495) - 443 (302–880) p < 0.0012nd trimester 2686 (2177–3204) - -

3rd trimester 4371 (3626–6081) - -

2 Progesterone, P4 (ng/ml) - 1.89 (0.56–3.38) - *p < 0.01, #p < 0.001, $p < 0.001

1st trimester 27.2 (16.3–37.4) - 5.83 (0.15–9.08) p < 0.0012nd trimester 67.8 (43.1–82.3) - -

3rd trimester 128 (107–164) - -

3 P4/E2 - 2.34 (0.93–5.28) – *p < 0.001, #p < 0.001, $p < 0.001

1st trimester 18.4 (13.5–30.1) - 11.01 (0.25–22.2) p < 0.012nd trimester 21.3 (12.2–36.2) - -

3rd trimester 29.2 (17.3–37.7) - -

1st trimester (8–12 weeks of gestation), 2nd trimester (20–24 weeks of gestation), 3rd trimester (28–32 weeks of gestation)* Represents p value between 1st trimester and post-partum#Represents p value between 2nd trimester and post-partum$Represents p value between 3rd trimester and post-partum

Italic fonts indicate significant variables

Data are shown as the median (interquartile range)

Fig. 6 Correlation of serum levels of progesterone to estradiol ratio (P4/E2) with SP-A during the pregnancy and post-partum (a) and SP-D atearly stages of gestation (8–12 weeks) in normal pregnancy and missed

abortion, “MA” (b). p < 0.05 represents a significant correlation using theSpearmanmethod. SP-A, surfactant protein A; SP-D; surfactant protein D

Reprod. Sci.

emphasizes the importance of sustained levels of SP-A andSP-D in the normal pregnancy. Any dysregulation in the op-timal levels of collectins could be an indicator of dysregulatedplacental development.

Anti-cancer role of SP-A and SP-D provides an interestingperspective to the consistently decreased levels of these pro-teins in pregnancy. A substantial body of evidence suggeststhat the pathways involved in tumor progression are similar tothose activated in the developing human placenta to promotethe trophoblast invasion and angiogenesis [34]. SP-A has beenshown to inhibit lung cancer progression by controlling the

polarization of TAMs [47]. Exogenous SP-D, though at sig-nificantly higher concentrations (> 10 μg/ml) than the physi-ological serum levels during pregnancy, reduced the viabilityof a range of human cancer cell lines including Jurkat, Raji,THP-1, MCF-7, and AML14.3D10 [33]. In coherence withthese reports, we observed a significant decrease in viabilityand migration of HTR-8/SVneo cells in the presence of rfhSP-D suggesting that consistently decreased serum levels of SP-Din normal pregnancy could be contributing to the placentaldevelopment and establishment of pregnancy. We hypothe-size that further decrease observed in the maternal serum

Fig. 7 Localization of SP-D in HTR-8/SVneo cells and effect of exogenousrfhSP-D on HTR-8/SVneo cell viability and migration. (a)Immunofluorescence analysis demonstrates the presence of SP-D protein incytoplasm and cell membrane of HTR-8/SVneo cells (green) (v, vi).Trophoblast cell identity was confirmed by cytokeratin 7 (CK-7) staining(green) (ii, iii). Inset image is negative control stained with FITC taggedsecondary antibody. Scale bars = 20 μm. (b) Phase contrast image of HTR-8/SVneo cells (i); wound healing assay, artificial wound was created on thecell surface in untreated cells (ii, iv, vi) and treated cells (viii). Images werecaptured at 0 and 16 h of treatment, scale bars = 100μm.HTR-8/SVneo cellssupplemented with DMEM/F-12 containing 2% FBS was used as a positive

control (ii, iii); DMEM/F-12 containing 2%CSSwas used as amedia control(iv, v); DMEM/F-12 containing 2% CSS+ rfhSP-D buffer was used as avehicle control (vi, vii); and treated was DMEM/F-12 containing 2% CSS+ rfhSP-D (10 μg/ml) (viii, ix). (c) % of wound closure was calculated after16 h of treatment and represented as bar graphs. (d) Viability assay, HTR-8/SVneo cells were treated with rfhSP-D (10 μg/ml) for 16 h and 24 h. %viability was calculated and represented as bar graphs. Cells supplementedwith DMEM/F-12 containing 2% CSS served as media control andDMEM/F-12 containing 2% CSS + rfhSP-D buffer served as vehiclecontrol. Data represented as mean ± SEM. *p < 0.05, **p < 0.01,***p< 0.001 using one way ANOVA with Tukey’s post hoc test

Reprod. Sci.

SP-D levels of “MA” women at early stages of pregnancymay be a positive feedback response to facilitate the develop-ment of placenta. SP-D inhibited the trophoblast migrationplausibly via binding the epidermal growth factor recep-tor (EGFR) on trophoblast cells leading to interferencein EGF-mediated signaling essential for the trophoblastinvasion [48, 49]..

Steroid hormones are important to regulate the immunefunction during pregnancy. It is well known that E2 and P4do not act independently [50]. Instead, P4/E2 ratio is moreimportant than absolute levels of these hormones as an indi-cator of the hormonal environment appropriate for implanta-tion and thus for the establishment of pregnancy [50]. Weobserved that although both P4 and E2 levels increased sig-nificantly in the normal pregnancy, the mean P4/E2 ratio de-creased in the first trimester of pregnancy and remained sothroughout the pregnancy when compared with the meanP4/E2 ratio reported previously in the luteal phase of non-pregnant women [39–41]. We calculated the P4/E2 ratiosfrom the reported luteal phase E2 and P4 levels in womenundergoing IVF/ICSI cycle. Similar to our observation, thewomen with successful clinical pregnancy had significantlylower P4/E2 ratios on day 14 of embryo transfer comparedwith non-pregnant [51]. Importantly, P4/E2 ratio in the firsttrimester of women, who underwent missed abortion during 14–20 weeks of gestation in our study, was found to be significantlylower than the normal pregnant women. This finding is consis-tent with the previously reported decreased serum P4/E2 ratio atthe time of implantation in women who subsequently underwentspontaneous abortion in the first trimester [52].

It is known that there is dysregulated placental develop-ment and immune regulation in pregnant women who under-go miscarriage. As evident from our data, the women in MAgroup had significantly lower P4, E2 as well as P4/E2 ratiothan healthy pregnant women that could be a factor resultingfrom and/or leading to defective placental development. Theselower P4/E2 ratios may have resulted in significantly lowerthan optimal levels of SP-D in the MA group than healthypregnant women. This view is supported by the significantpositive correlation of P4/E2 ratio with the serum levels ofSP-D in the first trimester of “MA.” Earlier, we reported asignificant decrease in the serum levels of SP-A, SP-D, andtheir positive correlation with P4/E2 ratio at 10–20 weeks ofgestation in women with subsequent severe EOPE [17]. SP-Dhas two important roles at the feto-maternal interface as sug-gested by the consistent serum levels during normal pregnan-cy though at levels significantly lower than the non-pregnantwomen. Firstly, we hypothesize that SP-D levels are loweredto facilitate placental development based on the findings thatSP-D binds to EGFR and inhibits EGFR signaling. Secondly,and importantly, SP-D levels are maintained consistently dur-ing the normal pregnancy suggesting its critical role in immu-noregulation and clearance of apoptotic trophoblasts at the

feto-maternal interface. Hence, the significantly lower serumlevels of SP-D in the MA group, although would favor theplacental development but may result in immune dysregula-tion contributing to inflammation-mediated miscarriage.

Placenta is both a steroid responsive organ as well as a majorsource of progesterone and estradiol during pregnancy.Correlation of systemic levels of SP-D and P4/E2 ratio duringpregnancy and ‘MA’ prompted us to investigate the localizedhormonal regulation of SP-D. Hence, we analyzed SP-D tran-scripts in the HTR-8/SVneo cells treated with E2, P4 and P4/E2ratio (details provided in Supplementary Information). Similar toour previous observations in non-pregnant murine uterus [26],we observed a significant upregulation of SP-D transcripts in thepresence of E2 (10nM) and significant downregulation of SP-Dtranscripts in the presence of P4 (100nM).We then treated HTR-8/SVneo cells with both E2 and P4 together in an increasing P4/E2 ratio (10, 20, and 100). The chosen P4/E2 ratios of 10, 20, and100 were approximately similar to the ratios observed in serumof “MA,” normal pregnancy, and non-pregnant women, respec-tively. We observed a significant decrease in the expression ofSP-D transcripts with an increased P4/E2 ratio in HTR-8/SVneocells (Supplementary Fig. S3). Thiswas in contrast to the positivecorrelation observed betweenmaternal serum levels of SP-D andP4/E2 ratio. It is inferred that during pregnancy, both placentaland systemic levels of SP-D are regulated by the P4/E2 ratio in adifferential manner. Since SP-D is expressed by the epithelial celllining various organs, systemic levels of SP-D are not only con-tributed by the placenta but also by other organs such as the brain,heart, intestine, and pancreas [53]. Besides hormones, the sys-temic levels of collectins are reported to be influenced by otherpro-inflammatory factors.

Our observation of a significant increase in the serum levels ofSP-A post-partum is consistent with the previous reports (6.0–102.4 ng/ml post-partum vs 1.4–83.9 ng/ml during pregnancy)[23]. This observation strengthened the role of SP-A in parturi-tion, as suggested by various studies [18, 23, 54]. One of thepossible mechanisms for the elevated levels of SP-A post-partumcould be thewithdrawal of progesterone at the time of parturition,as indicated by the observed significant negative correlation ofSP-A levels with P4/E2 ratio during pregnancy and post-partum.

In contrast with the previous reports [22, 55], we did notobserve any significant alteration in the serum levels of MBLduring pregnancy and early first trimester in “MA” in ourstudy cohort. Different ethnicity could be a reason for thedistinct observations as the circulating levels of MBL areknown to be genetically regulated.

Our data involving serum levels of steroid hormones (E2,P4, and FT) during normal pregnancy and in MA is in coher-ence with the previous reports [4, 56–58]. Moreover, we reporta significant progressively increased P4/FT and E2/FT ratioduring normal pregnancy, and a significant downregulation ofboth P4/FT and E2/FT in women destined to undergo “MA.”Significant correlation of steroid hormones (E2, P4, and FT)

Reprod. Sci.

and their ratio (P4/E2, P4/FT, and E2/FT) with SP-D in “MA”suggests their potential utility as combined predictive markersfor “MA.”

Polymorphisms in the SP-A (SFTPA), SP-D (SFTPD) andMBL genes have been reported to affect their serum levels. Itis possible that polymorphisms in the SFTPA and SFTPDgenes are linked to the genetic predisposition to “MA.” Wealso acknowledge a relatively small number of cases of “MA”owing to the prospective study design and a significant loss tofollow-up during the study. However, the present study great-ly benefits from its prospective nature and inclusion of bothnon-pregnant and post-partum healthy women as controls. Inaddition, the study, for the first time, established the normalrange of levels of SP-A, SP-D, and MBL in the maternalserum during pregnancy in an Indian cohort which can be veryuseful for future research and clinical evaluation.

In conclusion, the study infers that the maternal serumlevels of SP-A and SP-D are significantly downregulatedand are consistently maintained until 32 weeks of gestationin the normal pregnancy. Dysregulated levels of SP-D, E2, P4,and P4/E2 ratio in the early first trimester may indicateimpending missed abortion, and hence, could be useful aspotential predictive markers of spontaneous abortion, inasymptomatic women. Most of the previous studies focusedon the prediction of spontaneous abortion in women withsymptoms (threatened abortions). The results of our studysuggest that it is evenmore important to screen normal healthyasymptomatic pregnant women and predict their risk for animpending “MA.” This would facilitate counseling of suchhigh-risk pregnant women during early stages of pregnancyand administer potential treatments.

Acknowledgments We thank Dr. Smita Mahale, Director, ICMR-National Institute for Research in Reproductive Health, for providingthe financial support and experimental facilities to carry out the study(Accession No. RA/636/05-2018). Authors are thankful to Dr. ShahinaBegum, ICMR-NIRRH for her valuable suggestions regarding the appro-priate statistical tests for the study. We are grateful to Mr. VaibhavShinde, graphics expert, NIRRH, for his help in improving the resolutionof figures. KK acknowledges the ICMR-NIRRH and ICMR for providingJunior and Senior Research Fellowships.We are grateful to all the womenparticipants for their contribution to the study.

Funding Information The authors received financial support fromICMR-National Institute for Research in Reproductive Health to carryout the study (Accession No. RA/636/05-2018). SA was funded byResearchers Supporting Project (RSP-2020/26) King Saud University,Riyadh, Saudi Arabia.

Compliance with Ethical Standards Serum samples were ob-tained with written informed consent of the participants, and the protocolwas approved by the Institutional Ethical Committee of Indian Council ofMedical Research-National Institute for Research in Reproductive Health(ICMR-NIRRH), Mumbai (No. 276/2015), Ethics Committee forResearch on Human Subjects of DY Patil Hospital, Nerul, NaviMumbai (PDYPMC/Ethics/2015), and Wadia Maternity Hospital, Parel,Mumbai (IEC-NWMH/AP/2015/003-version 2).

Conflict of Interest The authors declare that they have no conflict ofinterest.

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