ENDOMETRIOSIS AND IMPAIRED PLACENTATION: A PROSPECTIVE COHORT STUDY COMPARING PLGF IN PREGNANCIES OF PATIENTS WITH AND WITHOUT ENDOMETRIOSIS

I ntroduction. Endometriosis is a chronic condi- tion that affects 10% of women of childbearing age. Pregnancies associated with endometriosis have addi- tional risks, such as preeclampsia, preterm birth, and foetal growth restriction. T he objective of the study was to investigate if en- dometriosis affects placental function during pregnan- cy, measured by placental growth factor (PLGF) when compared to unaffected patients.

and methods. In this prospective cohort study , we enro lled pregnan t women wi th endome- triosis according to laparoscopic findings, who were matched for body mass index and parity in a 1:1 ratio with healthy patients. Pregnant women with endome- triosis were compared to non-endometriosis patients and assessed for PLGF and pregnancy-associated ORIGINAL PAPER ENDOMETRIOSIS AND IMPAIRED PLACENTATION: A PROSPECTIVE COHORT STUDY COMPARING PLGF IN PREGNANCIES OF PATIENTS WITH AND WITHOUT ENDOMETRIOSIS Cosmina R. COSTACHE1,2 , Draga M. MANDI1,3 , Mariana C. COSTACHE OUTAS2, Traean BURCOS1,3 1 Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania 2 Integrated Ambulatory of Coltea Clinical Hospital, Bucharest, Romania 3 General surgery Department of Coltea Clinical Hospital, Bucharest, Romania Received 23th May, Accepted 07th June 2024 https:/ / doi.org/10.3 1688/ ABMU.2024.59 .2.04 Address for correspondence: Cosmina R. COSTACHE Bulevardul Ion C. Bratianu 1, Bucuresti 030167, Integrated AmbulatoryEmail: [email protected] Draga M. MANDI Bulevardul Ion C. Bratianu 1, Bucuresti 030167, General Surgery Departament Email: [email protected], Phone +40724636055 Endometriosis and impaired placentation: a prospective cohort study comparing PLGF in… – COSTACHE et al 168 / vol. 59, no. 2

Around 10% of women of childbearing age have endometriosis, a chronic, hormone-dependent illness that res ults in infertility, painful periods, and pelvic pain 1. Research has also connected endometriosis to adverse pregnancy and delivery outcomes, including elevated risks of small for gestational age newborns, hy- pertension, and pre-eclampsia as a result of decreased deep placentation brought on by abnormalities in the uterus and surrounding tissues 2. Over the last 20 years, research has highlighted that a combined screening test developed by the Fetal Medicine Foundation (FMF) is the most effective at detecting these complications when compared to other tests. This screening includes analyzing maternal demographic data and specific bio- markers, with the uterine artery Doppler measurement of the pulsatility index being particularly crucial 3. This

has proven to be a reliable early detector of pre-eclampsia, fetal growth restrictions, and small for gestational age infants, and is now integrated into standard obstetric practice 4. Prior research comparing pregnant women with and without endometriosis revealed no appreciable changes in uterine artery Doppler readings or levels of pregnancy-associated plasma protein-A (PAPP-A), with the exception of extreme instances. These stud- ies, however, did not account for gestational age or enceintes atteintes d’endométriose selon les résultats laparoscopiques et ont été comparés pour l’indice de masse corporelle et la parité dans un rapport de 1:1 avec les patients en bonne santé. Les femmes enceintes atteintes d’endométriose ont été comparées à des pa- tients sans endométriose et évaluées pour le PLGF et le PAPP-A à 11-14 semaines de gestation. La prééclampsie a été notée au troisième trimestre. Résultats. L’étude a révélé que les patients atteints d’endométriose avaient des taux de PLGF significati- vement plus faibles que ceux des contrôles (p=0,003). Dans notre étude, l’endométriose a été associée à une diminution des taux de PLGF et à une prééclampsie (p=0.026). Conclusions. Selon ces résultats, l’endométriose est associée à une perfusion placentaire tardive clinique- ment mesurable altérée. Il est recommandé que les pa- tients atteints d’endométriose aient un suivi plus étroit pour mieux prédire et prévenir les résultats périnatals et de grossesse dus à une perfusion altérée du placenta. Mots-clés: endométriose, placenta, artère utérine, PLGF. plasma protein A (PAPP-A) at 11-14 weeks of gestation. Preeclampsia was noted in the third trimester. Results. The study found that patients with endome- triosis had significantly lower PLGF levels compared to control (p = 0.003). In our study, endometriosis was associated with decreased PLGF levels and preeclamp- sia (p = 0.026). Conclusions. According to these results, endometriosis is associated with a clinically measurable impaired late placental perfusion. It is recommended that patients with endometriosis must have a closer follow up for bet- ter prediction and prevention of perinatal and pregnan- cy outcomes due to impaired perfusion of the placenta.

endometriosis, placenta, uterine arteries, PLGF. List of abbreviations: Beta HCG = -human chorionic gonadotrophin BMI = body mass index CRL = crown rump lenght DE = deep endometriosis FGR = fetal growth restriction FMF = Fetal Medicine Foundation GA = gestational age IQR = interquartile ISUOG = International Society of Ultrasound in ob- stetrics and Gynecology IVF = in vitro fertilization LMP = last menstrual period MOM = multiples of median OMA = ovarian endometrioma PAPP-A = pregnancy-associated plasma protein A PLGF = placental growth factor PE = preeclampsia PTB = preterm birth SD = standard deviation UtA-PI = pulsatility index of uterine artery VEGF = vascular endothelial growth factor Archives of the Balkan Medical Union June 2024 / 169 take into account additional influencing variables such as in vitro fertilization (IVF) conception 5,6. PLGF, a member of the vascular endothelial growth factor (VEGF) family, amplifies the angiogenic effects of VEGF7. This protein, which is glycosylated and exists as a dimer, is well-known for its ability to promote the formation of new blood vessels and the development of trophoblasts in the maternal decidua. It also plays a critical role in the differentiation and invasion of trophoblasts. The PLGF gene is located on chromosome 14q.14 and generates four PLGF isoforms. PLGF-1 and –2 are the main isoforms seen throughout pregnancy and are highly expressed in vil- lous trophoblast cells. PLGF-2 and PLGF-4 vary from PLGF by possessing extra heparin-binding domains, even though PLGF still retains 53% of its structural similarity with VEGF-A 8. Throughout pregnancy, a secondary phase of spi- ral artery remodelling occurs between 16 and 18 weeks of gestation, which corresponds with an increase in PLGF expression in the placenta throughout the second trimester. It is believed that PLGF causes angiogenesis to change from branching to non-branching starting in week 25, which helps the placental capillary network that is intended for low resistance to grow. PLGF levels start to drop towards the end of pregnancy and peak between weeks 29 and 32. Placental hypoxia from uter- oplacental ischemia specifically suppresses the produc- tion of PLGF in trophoblastic cells, whereas non-troph- oblastic cells upregulate PLGF expression in response to damage.While non-trophoblastic cells respond to damage by upregulating PLGF expression, uteroplacen- tal ischemia-induced placental hypoxia specifically sup- presses the production of PLGF by trophoblastic cells 9. Instead of just treating preeclampsia (PE) pa- tients who show with late-onset clinical symptoms, doctors can take proactive steps to prevent and screen for PE by identifying high-risk patients early on. Interestingly, decreased PLGF levels can be seen as early as 11–13 weeks of gestation, providing a window of opportunity for early first-trimester screening. THE OBJECTIVE OF THE STUDY was to assess if there is any difference in PLGF levels in women with and without endometriosis throughout the first trimester. By measuring alterations in late uterine perfusion, endometriosis raises the likelihood of placental mal- function; this understanding might assist elucidate this involvement.

Study design and setting This was an observational, monocentric, pro- spective cohort study carried out from January 2019 to January 2022 in Bucharest, Romania. Group of study The study included only singleton pregnancies that were diagnosed with endometriosis and verified with laparoscopic surgery prior to pregnancy. The endometriotic lesions were classified according to their location as either ovarian endometriomas, deep endometriosis, or a mix of both. Patients with adeno- myosis, numerous pregnancies, foetal abnormalities, uterine pathology, pre-existing maternal comorbidi- ties such as cardiovascular, liver, or renal problems, diabetes, coagulation disorders, and autoimmune dis- eases were excluded 10. Surgical and histological data were examined during the inclusion of patients. The control group consisted of randomly chosen pregnant women who had no previous history of en- dometriosis. The criteria for selecting controls were the absence of a previous diagnosis of endometriosis, a normal transvaginal ultrasound before pregnancy, and no history of considerable menstrual discomfort, painful intercourse, or persistent severe pelvic pain. Controls were paired with cases in a one-to-one ratio. Data collection The data gathering followed the ethical guide- lines outlined in the Declaration of Helsinki 8. The determin ation of pregnancy dates relied on the last menstrual period (LMP) for natural conceptions, whereas for IVF conceptions, it was determined as 19 days before blastocyst transfer or 1 7 days before cleavage-stage embryo transfer. The gestational age (GA) was determined during the first trimester us- ing ultrasound measurements of the crown-rump length (CRL), following the recommendations set by the Foetal Medicine Foundation (FMF) and the International Society of Ultrasound in Obstetrics and Gynaecology (ISUOG) 10. The Doppler ultrasonography was used to meas- ure the uterine artery pulsatility index (UtA-PI) throughout the 11-14 weeks of pregnancy, following the guidelines established by the FMF and ISUOG 11. The UtA-PI was determined by subtracting the peak systolic velocity from the end diastolic velocity and t hen dividing the result by the time-averaged veloc- ity. Measurements from both sides were averaged to provide an overall mean, which was then compared to a reference value 3,10 . All Doppler ultrasound assessments were per- formed by certified experts using high-end Voluson E8 and Voluson E10, GE HealthCare Technologies, USA devices fitted with multi-frequency convex transabdominal transducers. The data collection encompassed initial ma- ternal parameters, such as age, BMI, parity, concep- tion technique, and smoking status 6, first trimester ultrasound characteristics and placental hormone Endometriosis and impaired placentation: a prospective cohort study comparing PLGF in… – COSTACHE et al 170 / vol. 59, no. 2 biochemistry values. The collected data consisted of measurements of Crown-rump length (CRL), serum levels of beta-hCG and PAPP-A, and PLGF translated into multiples of the median (MoM) 12. Outcome The result of this study was the computation of the risk of preeclampsia throughout the initial three months of pregnancy. The scores were calculated us- ing reference equations based on previously published normal ranges. This allows for the evaluation of aber- rations in placental hormone readings compared to recognised norms throughout these pregnancies. Statistical analysis The statistical analysis started by doing the Shapiro-Wilk test to see if the continuous variables conformed to a normal distribution. Variables that were normally distributed were presented as the mean ± standard deviation (SD), while variables that did not follow a normal distribution were represented using the median and interquartile range (IQR). The categorical variables were shown as both absolute val- ues and percentages (%). To ensure comparability between the data from cases and controls, appropriate tests were utilised based on the characteristics and distribution of the data. The Student t-test was used for quantitative var- iables that exhibited a normal distribution, whereas the Wilcoxon signed-rank test was used for variables that did not exhibit a normal distribution. The se- lection of either Pearson’s Chi-square test or Fisher’s exact test was determined by the anticipated frequen- cies for categorical variables. We did a research to examine the relationship between endometriosis and PLGF Z-scores. We used minimal deviance analysis to identify the most precise connection and variance functions. Subsequently, we employed conventional goodness-of-fit tests to choose the optimal multivariable model. The findings also displayed the mean marginal effects for all variables in the model, along with 95% confidence intervals. The statistical analyses were conducted using STATA version 17. For all studies, a p-value less than 0.05 was considered to have statistical significance.

Study population A total of 12 individuals diagnosed with en- dometriosis were included in the research. Out of these cases, 10 (83.3%) had ovarian endometriomas (OMA), 6 (50%) had deep endometriosis (DE), and 2 (16.6%) had both OMA and DE localizations. Out of the 12 patients who had ovarian involvement, bilateral endometriomas were seen in 2 cases, ac- counting for 16.6% of the total. The study cohort consisted of 12 cases of endo- metriosis, which made up 50% of the overall group, and 12 controls, which accounted for the remaining 50%. The control group exhibited no signs of the ill- ness and were selected to match the patients in terms of parity and body mass index (BMI). Baseline characteristics and univariable analysis Table 1 presents the fundamental features of both endometriosis patients and controls. Based on the comparison with the matched controls, there were no notable disparities seen in BMI and parity (specifically, nulliparous vs. parous). Moreover, the mother age, cigarette smoking status, and conception technique (spontaneous versus in vitro fertilisation) were similar in both groups. 87.5% of cases were relat- ed to IVF cycles that utilised frozen-thawed embryo transfers (95% confidence interval of 74.4–94.4%). Table 2 provides a complete evaluation of ultra- sonography (US) factors throughout the first trimes- ter for both patients and controls. T his table presents a comprehensive comparison of several ultrasound measures obtained during the first trimester for both cases and controls, along with the statistical signifi- cance of the observed discrepancies. There were no notable variations in the Z-scores of the uterine artery pulsatility index (UtA-PI) between the two groups during the first trimester. During the first trimester, the MoM PlGF levels were substantially lower in the group with endometriosis (median 1.098, interquar- tile range 0.37 to 1.75) compared to the control group (median 1.56, interquartile range 1.01 to 1.85), with a p-value of 0.003. No significant variations were seen in the serum biomarkers (MoM PAPP-A and MoM free -hCG) or in the standardisation of crown-rump length (CRL) throughout the first trimester. In ad- dition, there was a slightly increased occurrence of PE in the observed cases, but this difference was not statistically significant.

This study found that pregnant patients with en- dometriosis had a greater incidence of PE in the third trimester compared to patients without endometriosis. However, there were no changes in the UtA-PI Z-scores during the first trimester between the two groups. We would want to explore potential pathways based on our findings. Typically, PLGF levels rise as pregnancy advances, indicating the development of the placenta and the conversion of spiral arteries into uteroplacental arteries. The increase in PLGF empha- sises the significance of ongoing maternal hemody- namic adaptations throughout the third trimester. Archives of the Balkan Medical Union June 2024 / 171 Endometriosis-related fibrosis can impact the performance of peripheral arteries by modifying en- dothelial function and raising vascular stiffness. The fibrotic entrapment of pelvic blood arteries may re- sult in the hardening of artery walls and decreased vascular flexibility, which might hinder the usual hemodynamic adaptations observed during the later stages of pregnancy. Therefore, it is only in the third trimester that the increased resistance in uterine arteries in endometriosis compared to controls be- comes evident, as a result of concurrent alterations in maternal hemodynamics 13. The decrease in uterine artery pulsatility index (UtA-PI) during pregnancy is also affected by increas- ing oestrogen levels, which typically encourage vasodi- lation. However, in cases of endometriosis, the pelvic region frequently exhibits a condition of excessive oes- trogen, which can lead to an abnormal reaction of the uterine blood vessels to oestrogen during pregnancy. The hormonal activity, which involves the interac- tion between oestrogen and progesterone, is precisely regulated by the controlled expression of steroid re- ceptors, chaperone proteins, and signalling pathways. Disruptions in these pathways in endometriosis might

in incorrect interactions between the chorion and decidua in the latter stages of pregnancy 13. Recent studies have also brought attention to possible problems associated with deep implantation of the pla centa in endometriosis, which is connected to structural and functional abnormalities in the lining of the uterus and the muscular layer of the uterus. The presence of endometrial tissue in abnor- mal locations in endometriosis results in the produc- tion of excessive amounts of cytokines that promote inflammation and fibrosis. This can interfere with the normal functioning of the immune system and blood vessels, which are crucial for the healthy inter- action between the placenta and the uterine lining 14. As a consequence, complications such as PE, foetal growth restriction, and preterm birth may arise. During normal pregnancies, spiral arteries undergo a process of remodelling, transforming into high-flow channels with low resistance. These transformed ar- teries play a crucial role in supporting the growth of the foetus 15. Pregnancies impacted by endometriosis may maintain the musculo-elastic composition of Table 1. Demographic characteristics of both cases of endometriosis and controls Variable Cases (n=12) Controls (n=12) p-value Maternal age, years 33.5±4.9 34.08±3.9 0.157 BMI, kg/m² 21.46 (15.8 to 28) 22.91 (19.1 to 26.7) 0.816 Smoking, % 3 (25%) 3 (25%) 0.656 Nulliparous, % 10 (83.3%) 9 (75%) 0.969 Spontaneous conception, % 3 (25%) 9 (75%) 0.738 IVF, % 9 (75%) 3 (25%) 0.738 Notes: – Data are presented as mean ± SD, median (IQR) or n (%). – Abbreviations: BMI – body mass index, IVF – in vitro fertilization. Table 2. Evaluation of ultrasonography and biochemical factors througho ut the first and third trimester in both patients with endometriosis and controls. Measurement Cases (n = 12) Controls (n = 12) p-value First trimester CRL, mm1 56.75±17.89 59 .26±7.36 0.441 IUGR- risk1 0.41±0.293 0.324±0.270 0.952 UtA PI2 1.435 (0.9 to 1.875) 1.61 (1.24 to 2.19) 0.715 MoM PLGF2 1.098 (0.37 to 1.75) 1,561(1.01 to 1.85) 0.003 MoM free -hCG2 0.89 (0.328 to 4.365) 1.22 (0.235 to 2.38) 0.31 MoM PAPP-A2 1.343 (0.271 to 2.129) 1.09 (0.25 to 2.12) 0.07 Third trimester PE3 50 % 25% 0.640 Notes: Data are 1 mean ± SD, 2 median (IQR) or 3 n (%). Abbreviations: CRL, crown-rump length; IUGR- intrauterine g rowth restriction; UtA PI, uterine artery pulsatility index; MoM, multiples of the normal median; free -hCG, free -human chorionic gonadotrophin; PAPP-A, pregnancy-associated plasma protein A; PLGF, placental growth factor, PE- preeclampsia Endometriosis and impaired placentation: a prospective cohort study comparing PLGF in… – COSTACHE et al 172 / vol. 59, no. 2 these arteries, resulting in heightened resistance to blood flow and complications with the development of the placenta. This study highlights the significance of in- creased uterine artery impedance during the third tri- mester as an indicator of placentation issues in women with endometriosis. This is associated with a higher likelihood of negative outcomes for both mother and baby. Although normal UtA-PI is observed in the first trimester, placental malfunction can advance from a subclinical stage to a clinically relevant condition throughout the later stages of pregnancy. Recent meta-analyses and systematic reviews have shown that endometriosis is linked to a range of adverse obstetric and neonatal outcomes. These diffi- culties encompass heightened chances of miscarriage, preterm birth (PTB), placenta previa, and newborns with small-for-gestational-age, among other potential issues. The presence of robust evidence is hindered by the inconsistency in diagnostic criteria and catego- rization systems for endometriosis, making it difficult to interpret these findings consistently. This research also confirms that increasing ma- ternal age affects PLGF in the first trimester, with older women facing multiple adverse pregnancy out- comes due to generally decreased vascular compli- ance and cardiovascular adaptations. Furthermore, in IVF, especially when using frozen-thawed embryo transfers, has a considerable impact on third-trimes- ter uterine artery pulsatility indices (UtA-PIs), which is consistent with findings from earlier studies. While this study’s conclusions are robust, some

include the reliance on ultrasound for diagnosing deep and ovarian endometriosis and the generalizability of the results, primarily applica- ble to advanced stages of the disease. Nonetheless, the study’s strengths include stringent matching for known confounders, high-quality and consistent ul- trasound methodology, and a robust statistical analy- sis, enhancing the validity of our findings.

Endometriosis is associated with a significant decrease in placental perfusion during the third tri- mester of pregnancy, as indicated by PLGF Z-scores (95% CI 0.01-3.56, OR 0.23) that are 30% lower com- pared to unaffected individuals. This limitation may arise from fibrosis associated with endometriosis, which alters the vascular response to oestrogen and progesterone, as well as the inflammatory conditions in the pelvic area. Based on these discoveries, it is advisable to suggest increased surveillance for these individuals. This would need regular well-being and growth scans between weeks 28 and 36 of pregnancy, to identify or prevent problems that may occur be- cause of abnormal placental development. Additional investigation is required to elucidate the mechanism by which reduced blood flow in the placenta, which is linked to endometriosis, might result in difficulties during pregnancy and childbirth. The ultimate goal is to improve the outcomes of pregnancy for women affected by this condition. Aknowledgements: We would like to express our sincere gratitude to Alina Ursuleanu for her meticulous verification of the ob- stetrical outcomes, which was crucial for ensuring the rigor of this study. We would like to express our appreciation to Valentin Vl ădescu for his exceptional ability to manage the analytical data and conduct statistical analysis, which significantly enhanced our research findings. In addition, we would like to recognize the group of anesthesiologists, headed by Leti ția Coriu, whose specialized knowledge was essential in carrying out the surgeries that played a pivotal role in this study. Their combined contributions were es- sential for the success of this project.

1. Vigano P, Candiani M, Monno A, Giacomini E, Vercellini P, Somigliana E. Time to redefine endometriosis including its pro-fibrotic nature. Hum Reprod. 2018;33(3):347–52. 2. Smolarz B, Szy łło K, Romanowicz H. Endometriosis: epide- miology, classification, pathogenesis, treatment and genetics (review of literature). Int J Mol Sci. 2021;22(19). 3. Salomon LJ, Alfirevic Z, Bilardo CM, et al. ISUOG practice guidelines: performance of first-trimester fetal ultrasound scan. Ultrasound Obstet Gynecol . 2013;41(1):102–13. 4. Benaglia L, Candotti G, Papaleo E, et al. Pregnancy out- come in women with endometriosis achieving pregnancy with IVF. Hum Reprod . 2016;31(12):2730–6. 5. Sotiriadis A, Hernandez-Andrade E, da Silva Costa F, et al. ISUOG Practice Guidelines: role of ultrasound in screen- ing for and follow-up of pre-eclampsia. Ultrasound Obstet Gynecol. 2019;53(1):7–22. 6. Poon LC, Shennan A, Hyett JA, et al. The International Federation of Gynecology and Obstetrics (FIGO) initia- tive on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention. Int J Gynaecol Obstet. 2019;145 Suppl(Suppl 1):1–33. 7 . Goteri G, Altobelli E, T ossetta G, et al. High tempera- ture requirement A1, transforming growth factor beta1, phosphoSmad2 and Ki67 in eutopic and ectopic endo- metrium of women with endometriosis. Eur J Histochem. 2015;59(4):2570. 8. Creswell L, O’Gorman N, Palmer KR, da Silva Costa F , Rolnik DL. Perspectives on the use of placental growth fac- tor (PlGF) in the prediction and diagnosis of pre-eclamp- sia: recent insights and future steps. Int J Womens Health. 2023;15:255–71. 9. Lessey BA, Young SL. Homeostasis imbalance in the endometrium of women with implantation defects: the Archives of the Balkan Medical Union June 2024 / 173 role of estrogen and progesterone. Semin Reprod Med . 2014;32(5):365–75. 10. Bhide A, Acharya G, Bilardo CM, et al. ISUOG practice guidelines: use of Doppler ultrasonography in obstetrics. Ultrasound Obstet Gynecol. 2013;41(2):233–9. 11. Gómez O, Figueras F, Fernández S, Bennasar M, Martínez JM, Puerto B, et al. Reference ranges for uterine artery mean pulsatility index at 11-41 weeks of gestation. Ultrasound Obstet Gynecol. 2008;32(2):128–32. 12. Akolekar R, Zaragoza E, Poon LCY , Pepes S, Nicolaides KH. Maternal serum placental growth factor at 11 + 0 to 13 + 6 weeks of gestation in the prediction of pre-eclampsia. Ultrasound in Obstetrics and Gynecology. 2008;32(6):732–9. 13. Horton J, Sterrenburg M, Lane S, Maheshwari A, Li TC, Cheong Y. Reproductive, obstetric, and perinatal out- comes of women with adenomyosis and endometriosis: a systematic review and meta-analysis. Hum Reprod Update . 2019;25(5):592–632. 14. Lalani S, Choudhry AJ, Firth B, et al. Endometriosis and adverse maternal, fetal and neonatal outcomes, a systematic review and meta-analysis. Hum Reprod. 2018;33(10):1854–65. 15. Berlac JF, Hartwell D, Skovlund CW, Langhoff-Roos J, Lidegaard Ø. Endometriosis increases the risk of obstetri- cal and neonatal complications. Acta Obstet Gynecol Scand . 2017;96(6):751–60.