Characterisation of serum progesterone and progesterone-induced blocking factor (PIBF) levels across trimesters in healthy pregnant women

Progesterone-induced blocking factor (PIBF), which plays an important role in maintaining healthy pregnancies, has shown great promise as a prognostic biomarker for threatened miscarriage. To better characterise the physiological trends of progesterone and PIBF, we analysed serum progesterone and PIBF concentrations in healthy non-pregnant and pregnant women across trimesters. We saw increasing concentrations of progesterone and PIBF in pregnant women with advancing trimesters. The serum progesterone and PIBF percentiles across gestational age in healthy pregnancies can be used as a guide for the formulation of reference ranges. We also demonstrated a significant positive correlation between progesterone and PIBF levels. This study demonstrates increasing progesterone and PIBF concentrations in later trimesters and underscores the importance of progesterone and PIBF in healthy pregnancies. Characterisation of progesterone and PIBF across gestational age in healthy pregnant women may help to prognosticate pregnancy viability and support further research into the importance of progesterone and PIBF in the maintenance of healthy pregnancies.

modulating NK cell activity and thereby ensuring healthy pregnancies 18 . PIBF also helps to inhibit phospholipase A2 which decreases the synthesis of prostaglandins from arachidonic acid, contributing to decreased IL-12 production and cytotoxic NK activity 1,19 . PIBF may also play critical roles in controlling trophoblast invasion through the suppression of pro-invasive genes 20,21 . progesterone and progesterone-induced blocking factor (piBf) are potential promising biomarkers for predicting pregnancy viability. Spontaneous miscarriage, occurring in 15 to 20% of all pregnancies, is the most common cause of vaginal bleeding in early pregnancy 22,23 . Vaginal bleeding affects a significant proportion of women (16% to 25%) in the first trimester of pregnancy 24 . The presence of vaginal bleeding is associated with a 2.6 times higher incidence of fetal loss 25 , and causes substantial anxiety and stress for the expectant mother 24,26 .
Threatened miscarriage is defined as vaginal bleeding with a closed cervix, in the first 20 weeks of a viable intrauterine pregnancy 22 . Accurate prediction of the outcome of threatened miscarriage in women is important in identifying women at high risk of eventual miscarriage and for providing timely advice and care to this high risk group. A plethora of miscarriage biomarkers, including human chorionic gonadotropin (hCG), progesterone, cancer antigen 125 (CA-125), inhibin A, activin A, pregnancy-associated plasma protein-A (PAPP-A) and kisspeptin have been proposed as tools to assess pregnancy viability [27][28][29] .
Many studies have shown that low serum progesterone is associated with threatened miscarriage. Our research group has validated the use of a serum progesterone cut-off value of <35 nmol/L in predicting subsequent miscarriage in women presenting with threatened miscarriage 6,30 . Given progesterone's importance in maintaining healthy pregnancies, and PIBF's pivotal role in maintaining a favourable immune environment for the semi-allogeneic fetus as a downstream effector protein of progesterone, both progesterone and PIBF are promising biomarkers for predicting pregnancy viability.
Reference ranges for progesterone and progesterone-induced blocking factor (piBf) are essential to supporting the use of progesterone and piBf as biomarkers for predicting pregnancy viability. There is currently no generally accepted reference range, threshold or benchmark for normal serum PIBF levels across trimesters relative to serum progesterone in healthy pregnant women. The primary aim of this study is to characterise normal serum progesterone and PIBF values with advancing trimesters. This would enhance the potential of progesterone and PIBF in predicting pregnancy viability and aid in the development of the use of progesterone and PIBF levels as part of a resource saving and easy-to-use risk assessment tool for the identification and management of pregnant women at risk of miscarriage.

Results
Participants. 183 women met the inclusion criteria and participated in the study. Of this group, 46 healthy non-pregnant women, and 47, 48 and 42 healthy pregnant women in the first, second and third trimesters respectively were recruited. All pregnant participants had successful term deliveries without complications.
Maternal characteristics. Demographic variables collected from the participants (gestational age by ultrasound scan at recruitment, maternal age, body mass index (BMI), history of previous miscarriage, current smoker, and current alcohol drinker) were compared across the 4 subgroups (Table 1). Continuous variables were analysed with one-way ANOVA, while categorical variables were analysed using the chi-square test. Gestational age by ultrasound scan was significantly different among the 4 subgroups of participants, demonstrating the effective trimester segmentation of subgroups. Maternal age, BMI and history of previous miscarriages were comparable across the subgroups supporting the effectiveness of randomisation, while the proportion of current smokers and the proportion of current alcohol drinkers were predictably negligible in the healthy pregnant group relative to the non-pregnant group.
Linear regression of ln(PIBF) against ln(progesterone) revealed a significant positive linear relationship between the two variables with equation y = 0.764 x + 3.19 (r 2 = 0.474, p < 0.0001) and Pearson correlation coefficient r = 0.688 (p < 0.0001) (Fig. 3). Data from the non-pregnant group were excluded from the linear regression, as our study sought to investigate the effect of increasing progesterone on PIBF in healthy pregnancies.
Correlation of serum progesterone with BMI. A decrease in progesterone was seen with increasing BMI in pregnant women in the first trimester (Fig. 4). This relationship did not extend beyond the first trimester, and was not discerned in pregnant women in the second and third trimester (data not shown). There was no discernable relationship between PIBF and BMI (data not shown).

Discussion
Both serum progesterone and PIBF increased with advancing trimesters and increasing gestational age. The increase in PIBF concentration showed a significant positive correlation with the increase in progesterone concentration. Our research group has previously described a linear increase in progesterone levels from 5 to 13 weeks of gestation in healthy pregnancies 31 , and this study further supports the crucial role of progesterone in healthy pregnancies. This is one of the first prospective studies characterising the distribution of serum PIBF across trimesters in relation to progesterone in healthy pregnant women. There has been considerable interest in the development of reference ranges for serum PIBF across gestational age in relation to progesterone, which is PIBF's physiological precursor. The serum progesterone and PIBF percentiles across gestational age can guide the development of reference ranges for serum progesterone and PIBF across gestational age, and contribute to a prediction of pregnancy viability.
A decrease in progesterone with increasing BMI above the normal range was seen in healthy pregnant women in the first trimester. This is consistent with another study showing a significant correlation between maternal obesity and low serum progesterone (defined as <35 nmol/l) in the first trimester 32 . Probable mechanisms include the association of obesity with a secondary central decrease in luteinising hormone leading to reduced progesterone production and premature luteolysis 33 , the deleterious effects of adipocytokines on corpus luteum function 34,35 , and an effectively lower serum progesterone concentration secondary to the pharmacokinetic effect  of an increased progesterone distribution in adipose tissues due to the high lipid solubility of sex steroids 32 . These may support weight management and optimisation during the pre-conception period as possible means of increasing the chances of a successful pregnancy. This study affirms previous studies outlining the importance of PIBF in pregnancy. PIBF modulates crucial effector functions, including shifting cytokine synthesis and cytotoxic cell activity towards a more immunotolerant state 1,9 . CD4+ and CD8+ T cells have been found to respond to progesterone in a dose dependent manner,  www.nature.com/scientificreports www.nature.com/scientificreports/ causing marked changes in T-cell cytokine secretions at sites nearer to the fetus 36 . This dose dependent relationship between progesterone and PIBF is evidenced by the significant positive correlation between the two biomarkers seen in this study. The correlation is consistent with other studies that demonstrate increases in PIBF caused by progesterone 37 .
Characterisation of PIBF in healthy pregnancies across gestational age is anticipated to aid further investigations and assessments into its utility as a predictive tool for pregnancy viability. A potential advantage of PIBF over progesterone as a biomarker is that unlike progesterone, which undergoes reduction and glucuronidation before being excreted in different isomers, PIBF is excreted intact into the urine and can therefore be measured non-invasively 38 . In addition, unlike progesterone, which has been shown to decrease with increasing BMI, PIBF, a non-lipophilic protein, may not exhibit significant pharmacokinetic distribution in adipose tissues and hence may not be affected by BMI. Moreover, PIBF1 cDNA shares little amino acid homology with other known proteins 39 . These characteristics of PIBF enhance PIBF's appeal as a specific target to predict pregnancy outcomes.
PIBF concentrations, which are detectable and quantifiable in the serum and urine of pregnant women, may have utility for predicting pregnancy outcomes 5 . PIBF concentrations in urine samples have been shown to continually increase until the 37 th gestational week, after which PIBF concentrations fall 38 . One study suggested a trend indicating higher miscarriage rates when PIBF is absent at 3 to 5 weeks of seemingly normal pregnancies 40 , while another study demonstrated a positive correlation between PIBF and successful conception 41 . This study, along with previous studies, reaffirms the critical role of PIBF in maintaining healthy pregnancies, and postulates the potential of PIBF as an effective screening tool for threatened miscarriage.
PIBF levels can be compared to progesterone levels in pregnant women to assess the degree of progesterone-dependent immunomodulation, which may provide valuable prognostic and therapeutic options for the mother. If PIBF is abnormally low compared to progesterone, it may indicate relative lymphocytic insensitivity to progesterone through impaired receptor stimulation or defective PIBF production in progesterone receptor positive lymphocytes, even in the presence of progesterone. The downregulation of the progesterone receptors and their downstream effector PIBF has been described in women with preterm deliveries, shorter gestational periods and negative pregnancy outcomes, and has been correlated with a dominant Th1 and pro-inflammatory state 42 . Progesterone concentrations at the extremities of the body are lower than concentrations at the maternal-fetal interface, which may enhance the importance of lymphocytic sensitivity to progesterone at the body's peripheries in determining the eventual immunomodulatory effects of progesterone 1,43 . Correlating the progesterone and PIBF levels in the body's peripheries may identify women at risk of miscarriage, and women who may benefit most from immunotherapy.
PIBF is recognised as a potential therapeutic agent or marker in lymphocyte immunotherapy for the reduction of miscarriage risk. Given that current research has pointed towards an active recognition and response to fetal antigens by the maternal immune system during pregnancy 44 , and the concomitant deviation of the maternal immune system towards that of tolerance as evidenced by the key role of regulatory T cells in healthy pregnancies 45 , lymphocyte immunotherapy may aid in supporting the development of immunological tolerance to the semi-allogeneic fetus. This therapy may be instrumental in treating recurrent spontaneous abortions, given that Th1 cytokines and NK cell over-activity are said to be major alloimmune drivers of this condition 46 . A meta-analysis has found that lymphocyte immunotherapy shows promise in improving in the live birth rate of women with unexplained recurrent spontaneous abortions 47 . Another study has demonstrated the increased lymphocytic expression of PIBF after lymphocytic immunisation using the male partner's lymphocytes, pointing towards the utility of PIBF as a biomarker or active agent in alleviating miscarriage risk 48 . The findings of this www.nature.com/scientificreports www.nature.com/scientificreports/ study may provide guidance in PIBF dosing and therapeutic drug monitoring, if needed in the formulation of a safety profile and the elucidation of mechanisms behind such treatments.
Beyond obstetrics, PIBF may be relevant in oncology. PIBF has been described to suppress the immune response towards tumour cells, has been reported to be a positive regulator of tumour spread in primary lung and ovarian cells, and has been found to be expressed in the cytoplasm and nucleus of choriocarcinoma and primary tumour cells 49 . Furthermore, PIBF has been found to be expressed in various cancers, including glioblastoma multiforme, astrocytomas, and leukaemias, contributing to uncontrolled tumour proliferation and potential evasion of immune surveillance [50][51][52] . PIBF has also been reported to downregulate E-cadherin expression, possibly interfering with cell-cell adhesion mechanisms and increasing extracellular matrix degradation by matrix metalloproteinase (MMP), thus contributing to cancer invasion 49,53 . Therefore, beyond PIBF's potential utility as a biomarker for predicting pregnancy viability, characterisation of normal PIBF values in healthy women may contribute to future research and development of PIBF as a potential cancer biomarker and therapeutic target used to monitor the degree of cancer control and augment targeted cancer therapy.
Despite the potential utility of progesterone and PIBF as biomarkers for predicting pregnancy viability, this study is limited by a relatively small sample size and its design as a cross-sectional study. Further studies, including longitudinal studies, must be conducted with larger sample sizes and in different populations to validate the reference ranges for progesterone and PIBF across gestational age. A previous pilot study had explored a "best subset" 3-factor model comprising of progesterone, fetal heart and BMI for the prediction of spontaneous miscarriage 6 . Future studies can formulate, evaluate and validate further predictive models for pregnancy viability that include progesterone and PIBF, along with other relevant demographic factors, imaging findings and laboratory markers. Additionally, current kits used for PIBF quantification exhibit high variability. Development of new kits with better sensitivity and affinity for PIBF will enable more accurate quantification of PIBF, and will pave the way for the wider use of PIBF.

conclusion
This study underscores the importance of progesterone and PIBF in healthy pregnancies, with increasing progesterone and PIBF concentrations seen in advancing trimesters and increasing gestational age. The characterisation of progesterone and PIBF levels across gestational age in healthy pregnancies and in healthy non-pregnant women, along with a significant positive correlation of progesterone and PIBF, can aid in the formulation of normal reference ranges for progesterone and PIBF, may help to prognosticate pregnancy viability, and can support further research into the importance of progesterone and PIBF in the maintenance of healthy pregnancies.

Patient recruitment. Approval from the SingHealth Centralised Institutional Review Board (CIRB Reference
Number: 2017/2431) was obtained before patient recruitment commenced from 01 June 2017 to 01 December 2018. All aspects of the study, including patient recruitment and informed consent, were performed in accordance with the Declaration of Helsinki and the requirements of the SingHealth Centralised Institutional Review Board.
In this prospective cross-sectional study, healthy pregnant women in the first, second or third trimester who are patients at the KK Women's and Children's Hospital, and healthy non-pregnant women in KK Women's and Children's Hospital who are present for pre-conception or gynaecological consultations and satisfy the inclusion and exclusion criteria were identified by their doctors. The patients were approached and recruited in person on their routine visits or consultations at the KK Women's and Children's Hospital, and had their written informed consent taken by the investigators. A target of 200 healthy women between the ages of 21 and 45 years were recruited, comprising of 50 women who are not pregnant, and 50 pregnant women each in the first, second and third trimester with a single intrauterine pregnancy. Pregnant women who have been diagnosed with inevitable miscarriage, or women who have been known to have a history of recurrent miscarriages (3 or more consecutive pregnancy loss before 24 weeks of gestation), or women who have pre-existing luteal phase deficiency or other forms of diagnosed progesterone deficiency, or women who are planning to terminate pregnancy, or women with uncontrolled medical conditions, or women who have been treated with progesterone for any reasons in this current pregnancy, or women who have previous episodes of pregnancy related per vagina bleeding in this current pregnancy which may indicate threatened miscarriage were excluded.
Covariates for analysis, including maternal demographic, health, obstetric and lifestyle factors, were collected through an investigator-administered questionnaire. All participants were contacted and their case notes were checked to verify their successful delivery.
Sample collection and quantification of progesterone and PIBF. 10 ml of blood, taken via venipuncture, was collected from participants to measure serum progesterone and PIBF levels at presentation. Blood was collected into plain tubes and centrifuged for 10 min at 3000 g within 2 hours of collection. Serum samples were then stored at −80 degrees Celsius until analysis.
Serum progesterone levels were measured in the hospital's clinical laboratory using the commercial Abbott ARCHITECT progesterone kit, which is a chemiluminescent microparticle immunoassay (CMIA), in accordance with the manufacturer's protocol.
Serum PIBF concentrations were determined by an enzyme-linked immunosorbent assay (ELISA) using the Cusabio PIBF ELISA kit (CSB-E12872h, Cusabio Co. Ltd., China), which is a competitive enzyme immunoassay, in accordance with the manufacturer's protocol. Serum samples from healthy non-pregnant women and healthy pregnant women in the first trimester were diluted 10 times from their original concentrations. Serum samples from healthy pregnant women in the second or third trimester were diluted 50 times from their original concentrations. All serum samples were diluted with phosphate-buffered saline (PBS). 50 μl of the diluted serum sample was added to the wells containing PIBF-specific antibodies and horseradish peroxidase (HRP)-conjugated PIBF, thereby initiating a competitive inhibition reaction by the PIBF-specific antibodies between PIBF in serum