Effects of intralipid infusions on anti‐trophoblast antibody (ATAb)‐activities in patients with recurrent pregnancy loss: An observational report

1 INTRODUCTION

1–2% of women suffer from recurrent pregnancy loss (RPL) and many of these miscarriages remain unexplained.1, 2 It has been postulated that immunologic disorders may be responsible for unexplained RPL and several studies point to close links between pregnancy loss and various forms of immunopathology.2-14 In light of the increasing evidence of immunologic disorders associated with RPL, several immunomodulatory therapies have been evaluated in various groups of RPL patients.15-20 Preliminary data suggest beneficial effects of intravenous intralipid infusions (IVIL) in women with a history of RPL16, 21-27 and various immunomodulatory mechanisms have been claimed for IVIL to explain such results.21, 22, 26-33 Nevertheless, the clinical effects of IVIL in RPL patients are controversial and, moreover a clear mode of action of IVIL has not been established.

Intralipid is a commercially available fat emulsion. It is a synthetic product composed of purified soybean oil, purified egg phospholipids, glycerol anhydrous and water (Fresenuis Kabi Canada Ltd. Toronto). The 20% intravenous fat emulsion is used as parenteral nutrition.23 Contraindications are disturbed metabolism of fat, liver insufficiency and hypersensitivity to egg, soya or peanut protein. Adverse reactions such as a rise in body temperature, shivering, nausea or headache are specified with an incidence of < 1% according to the manufacturer information (Fresenuis Kabi Canada Ltd. Toronto).

We have shown that a subset of patients with idiopathic RPL express increased anti-trophoblast antibodies (ATAb)-activity14 and these antibodies appear to mediate immune pathology. Several studies, including our own, indicate such antibodies may be harmful for pregnancies.7, 14, 20, 34, 35

In light of such potentially harmful effects of ATAb and potentially beneficial effects of IVIL in RPL patients, we studied ATAb-activities in RPL patients receiving IVIL during a consecutive pregnancy.

2 MATERIALS AND METHODS 2.1 RPL patients

Patients of our RPL clinic with a history of at least three consecutive recurrent miscarriages before 20th gestational weeks (gw) were screened for established causes of their RPL. Laboratory tests were done at least 8 weeks after the last pregnancy had ended, in average 17 weeks (median 17 weeks, interquartile range (IQR): 8–42 weeks). None of these patients had received transfusions of blood or blood products. RPL work up was done according to ESHRE guidelines1 and Rogenhofer et al.14, 17

Briefly, uterine anomalies, endocrine dysfunctions,36 autoimmune disorders, deficiencies in coagulation factors, inherited thrombophilias as well as fetal and parental chromosomal aberrations were excluded as described1-4, 14, 17 An antiphospholipid syndrome was ruled out according to the international consensus statement regarding the classification criteria for this syndrome.37 In addition, sera of patients were analysed for ATAb-activity according to our pre-studies.14

2.2 Biographic and pregnancy histories

Biographic and historic data as well as pregnancy histories of RPL patients and healthy women are summarized in Table 1.

TABLE 1. History, biographic and obstetric data of the RPL patients and healthy volunteers RPL patients with IVIL n = 10 RPL patients without IVIL n = 2 Healthy volunteers n = 2 Age years mean (min-max)

35.6

(33-38)

33.5

(32-35)

35.5

(33-38)

BMI

Mean (min-max)

22.5

(20.1-25.5)

22.0

(21.8-22.2)

22.7

(21.7-23.8)

Pregnancies

Mean (min-max)

4.1

(3-7)

3.5

(3-4)

2

(2-2)

Miscarriages

Mean (min-max)

3.8

(3-5)

3.5

(3-4)

0

Deliveries

Mean (min-max)

2

(0-2)

0

2

(2-2)

Primary RPL 7 2 0 Secondary RPL 3 0 0 First trimester RPL 8 2 0 First trimester + second trimester RPL 2 0 0 Number of miscarriages 3 4 1 0 4 4 1 0 5 2 0 0 Abbreviations: RPL, recurrent pregnancy loss; IVIL, intravenous intralipid infusion ; BMI, body mass index (Calculated as weight in kilograms divided by the square of height in meters); n, number. Primary RPL: no infant born either dead or alive after the 20th completed week of gestation and/or weighing more than 500 g before the series of miscarriages [WHO, 38]. Secondary RPL: at least one infant born either dead or alive after the 20th completed week of gestation or weighing more than 500 g before the series of abortions [WHO, 38]. 2.3 ATAb-activity

This was measured as described.14 Briefly, serum samples (20 μl) were incubated with suspensions of 105 JEG-3 cells at 4°C for 60 min, washed and treated with 1:10 diluted FITC-conjugated goat anti-human secondary antibodies (Dako, Hamburg, Germany) for 60 min in the dark. After subsequent washing, cell suspensions were analysed on a Beckman Coulter flow cytometer (Beckman Coulter EPICS XL serial number AB 01019) equipped with a 2.4 mW argon ion laser at an excitation wavelength of 488 nm14 Reactivities were measured in mean channel shifts (MCS) and relative ATAb-activities were calculated as a percentage from the ratio of differences between the reactivity of the test sample and a low reacting standard over the difference of high (a pool of 10 positive patient sera - defined as 100%) and low reacting standard (a blood group AB Rh-positive standard sample - defined as 0%) reactivity.14 Positive ATAb-activity was defined to be above the 95% confidence interval (CI) of 110 female healthy controls without miscarriages. According to this calculation, relative ATAb-activities of > 39% were positive.14

Patients with positive ATAb-activities and otherwise unexplained RPL were selected for this observational report.

2.4 Off label treatment with intravenous intralipid preparations (IVIL)

Between 2019 and 2020, we informed RPL patients with positive ATAb-activities and no established cause for their miscarriages, about the lack of established treatment approaches and a possibility of using off label IVIL during a consecutive pregnancy as an individual healing attempt8, 16, 27 Also, we informed them of our interest in studying potential effects of IVIL infusions on their ATAb-activity. This approach was reviewed and supported by the Human Investigation Review Board of the Ludwig-Maximilians University Munich (IRB No 68-16). Ten patients agreed to participate in this observational study and signed informed consent. After early positive pregnancy tests, corresponding to week five of gestation, these patients received their first IVIL infusions as recommended by the supplier: 100 ml Lipovenös 20% (Fresenius, Kabi, Germany) were diluted with NaCl to totally 500 ml volume.

IVIL were repeated every 3 weeks until 33th gw as described by Roussev et al. and Coulam et al.16, 22 During each IVIL infusion, a pre- and a post- IVIL blood sample was drawn and ATAb-activities were measured as described above.

2.5 Expectant management

Two ATAb-positive RPL patients preferred expectant management but agreed in monitoring of their ATAb-activities. Starting with positive pregnancy tests at week five of gestation, these patients were seen every 2 weeks for clinical and sonographic routine check-ups. Blood was drawn and serum analysis of ATAb-activities were performed.

2.6 Normal pregnancies of healthy ATAb-negative volunteers

ATAb-activities were also studied during the course of normal gestations in two healthy volunteers without a history of previous miscarriages or other pregnancy complications. Prior to their pregnancies, both had negative ATAb-activities (9% and 23%).

2.7 Compliance with ethical standards: Informed consent and Ethical approval

All procedures performed in this study involving human participants were in accordance with the ethical standards and the national research committee and with the 1964 Helsinki declaration and the later amendments or comparable ethical standards. This study was approved by the Human Investigation Review Board of the Ludwig-Maximilians University Munich (IRB No 68-16).

2.8 Statistics

Variables are described by numbers (n) and mean ± standard deviation.

For statistical analyses the Statistical Package for Social Sciences was performed (SPSS for Windows 25.0, (SPSS Inc., 1989–2019, Chicago, IL, USA).

Statistical inferences relied on a mixed linear model with a random intercept and a random time effect for each patient, and a compound symmetry covariance structure. The models were fitted using the MIXED procedure of the Statistical Analysis System SAS, release 9.04 for Linux (SAS Institute, Cary, NC, USA). Plevels < .05 were considered as statistically significant.

3 RESULTS

The set of data presented in this paper comprises 12 patients with RPL and positive ATAb-activities. Ten of these received off label IVIL from 5th to 33th gw and two of these preferred not to receive IVIL during pregnancy, but agreed in monitoring of their ATAb-activity. In addition, ATAb–activities were studied during normal pregnancies of two healthy ATAb-negative women without histories of miscarriage or other pregnancy complications, who agreed in serial ATAb analysis.

No adverse side effects such as flushes, drowsiness, nausea, vomiting or fever were observed in the context of IVIL applications.

3.1 Pregnancy progress, deliveries and neonatal outcome

In total, nine of the 10 pregnancies who received IVIL were uneventful and resulted in nine healthy newborns ≥37th gw. Six of the mature newborns were delivered spontaneously (37th - 42th gw) and three by cesarean section. One cesarean section was on maternal demand, another due to cephalopelvic disproportion (4135 g birth weight) and a third one was performed in the 35th gw because of amniotic infection and consecutive premature rupture of membranes (birth weight 2860 g). The mean gestational date of delivery was 38.1 gw with a mean birth weight of 3245 g (2860 g - 4135 g). No neonatal malformations were recorded. One of the ten RPL patients receiving IVIL suffered a missed abortion at 7+6 gw and a karyotype of this embryo revealed a trisomy 16 (47 XY +16).

The two RPL patients with positive ATAb-activity and expectant management suffered missed abortions at 6+3 and 7+4 gw, respectively. In both cases, embryonic karyotyping revealed an euploid fetus with 46 XX.

One of the two uneventful pregnancies of the healthy controls was delivered by a cesarean section in the 40th gw due to cephalopelvic dysproportion with a birth weight of 4050 g. The second control patient delivered her healthy baby spontaneously at 41th gw with a birth weight of 3900 g.

3.2 ATAb-activities and course of pregnancies

Prior to pregnancies, ATAb-activities of the ten PRL patients, that agreed to receive IVIL, ranged from 42% to 72% (mean 55.2%) and from 58% to 71% (mean 64.5%) in the two patients who opted observational management (Table 2). During consecutive pregnancies, mean ATAb-activity prior to the first IVIL infusion (5th gw) was 56.8% and after IVIL the mean activity dropped to 49.3%. Indeed, this effect of IVIL in suppressing ATAb-activity was observed after each IVIL infusion, accounting for a mean reduction of 4.48% per infusion and this suppression was highly significant (P < 0,0001). As an additional effect of repeated IVIL infusions, mean ATAb-activities in pre-treatment samples showed a decrease of 2% per IVIL treatment and this additional over time effect was also significant (P = 0,002). For all patients receiving IVIL, this resulted in ATAb-activities in the normal rage (i.e., below 39%) by week 25 of gestation and beyond (Figure 1). Six of these patients spontaneously delivered healthy babies at gestational weeks 37–42 and two patients were delivered by cesarian section.

TABLE 2. Data of anti-trophoblast-(ATAb)-activities [%] prior (pre) and after (post) each intravenous intralipid infusion (IVIL) during pregnancy ATAb-activities mean (min -max) % pre- and post IVIL p1-p10* Reduction of ATAb-activities % pre- and post IVIL ATAb-activities mean (min -max) % expectant management ATAb-activities mean (min -max) % healthy volunteers Prior to pregnancy 55.2 (42-72) 64.5 (58-71) 16.0 (9-23) 5th gw

Pre IVIL

Post IVIL

56.8 (34-98)

49.3 (19-78)

7.5 72 (62-82) 15.5 (8-23) 7th gw

Pre IVIL

Post IVIL

54.2 (27-93)

49.5 (21-84)

4.7 74 (64-84) 13.5 (10-17) 9th gw pre IVIL 53.6 (28-84) 7.6 12.0 (6-18) post IVIL 46.0 (20-77) 12th gw pre IVIL 50.3 (23-83) 7.0 12.5 (8-17) post IVIL 43.3 (16-67) 15th gw pre IVIL 45.4 (24-70) 4.4 12.5 (8-17) post IVIL 41.0 (17-66) 18th gw pre IVIL 43.6 (23-73) 4.8 12.5 (9-16) post IVIL 38.8 (21-59) 21th gw pre IVIL 38.6 (14-63) 5.2 12.0 (9-15) post IVIL 33.4 (8-50) 24th gw pre IVIL 36.4 (23-54) 3.8 11.5 (9-14) post IVIL 32.6 (23-39) 27th gw

pre IVIL

32.1 (22-38)

post IVIL 29.2 (16-33) 2.9 10 (8-12) 30th gw pre IVIL 27.3 (11-33) 2.5 10 (8-12) post IVIL 24.8 (10-35) 33th gw pre IVIL 23.8 (1-35) 3.0 9 (8-10) post IVIL 20.8 (1-34) P1-P10: RPL patients with positive ATAb-activity treated with IVIL; *P10 aborted in 7+6 (47XY + 16); recorded until 7th gw. Expectant management: RPL patients with positive ATAb-activity without IVIL. Abbreviations: RPL, recurrent pregnancy loss; IVIL, intravenous intralipid infusion; ATAb, anti-trophoblast antibodies; gw, gestational week; n, number. image

Mean relative anti-trophoblast-(ATAb)-activities [%] of ATAb-positive RPL patients. Mean ATAb-activities of post-IVIL are significantly lower than in pre-IVIL samples (P < 0,0001). In addition, IVIL-treated patients show significant decrease of ATAb-activities (P < 0,002), resulting in normal values at and beyond gw 25. The two RPL patients opting not to receive IVIL show high mean ATAb-activities that remain high until they lose their euploid pregnancies at gw 6+3 and 7+4. Mean ATAb-activities of the two healthy ATAb-negative controls (C1/C2) remain low until they deliver healthy term infants. Cut off for positive ATAb-activity: 39% (horizontal line). RPL: recurrent pregnancy loss, IVIL: intravenous intralipid infusion, gw: gestational week

One RPL patient was diagnosed with missed abortion at gestational week 7+6, 1 week after receiving her second IVIL treatment, and the karyotype of this embryo revealed a trisomy 16 (47 XY + 16).

The two RPL patients opting for observational management had ATAb-activities of 58% and 71% (mean: 64.5%) prior to and 62% and 82% (mean: 72%) at 5th week of their consecutive pregnancies. These ATAb-activities remained at that range at gestational week 7 and 8, respectively: 64% and 84% (mean: 74%) (Table 2, Figure 1). During their visit at 6+3 and 7+4, respectively, their pregnancies were diagnosed with no heartbeat and missed abortions. Consecutive curettage and embryonic karyotyping revealed euploid karyotypes in both cases (46 XX).

Two healthy volunteers with no history of miscarriages or other pregnancy complications agreed in monitoring their ATAb-activities: prior to their pregnancies, they had ATAb-activities of 9% and 23% (mean: 16%) und their activities remained in that range for the entire course of their pregnancies (Table 2, Figure 1). Both delivered healthy term infants.

4 DISCUSSION

We analysed ATAb-activities in ATAb-positive RPL patients that were offered off label treatment with IVIL during consecutive pregnancies: Our results indicate a highly significant reduction of ATAb-activities in post- versus pre- IVIL sera, accounting for an average decrease of 4,48% per infusion of IVIL. We do not know the mechanism of IVIL reducing ATAb-activity and this could involve interference with antibodies and/or antigens involved.

ATAb have been associated with RPL and appear to mediate immune pathology.14

Our previous study showed that ATAb in vitro decrease HCG- and progesterone production and we have suggested, this could be a mechanism, by which ATAb interfere with normal gestation.34 It might be interesting, if IVIL in vivo, by reducing ATAb-activities results in improved HCG- and progesterone production. This potential mechanism of action requires further research; however, this small observational study has not been designed nor powered to approach this potential mechanism of action. Indeed, we studied a somewhat heterogeneous group of ATAb-positive patients, consisting of primary and secondary RPL and patients with first trimester and/or second trimester RPL. Our previous study did not reveal any significant differences in ATAb-activities of women with primary or secondary uRPL,14 so that we suggest, summarizing these subgroups for the analyses of ATAb-reactivities and clinical outcomes is acceptable.

We are not aware of studies indicating an interference of lipid emulsions in general or IVIL preparations in particular with alpha-enolase or its antigenicity39 Indeed, we are not aware of any studies indicating effects of lipid emulsions with antibody-antigen interactions in general.

Moreover, after repetitive IVIL treatments we also observed a significant reduction ATAb-activities in consecutive pre-treatment samples, accounting for an average decrease of 2% per IVIL treatment. This continuous reduction of ATAb-activities resulted in normal pre- and post IVIL-values in all treated patients after gestational week 25.

In contrast, the two RPL patients who opted not to receive IVIL showed high ATAb-activities that appeared to further increase, until losing their early pregnancies.

We do not know, if decrease of ATAb-activities is a result of IVIL and its repetitive administration or due to the progressing gestational age. With half-lives of infused intralipids reported in the range of only few hours40, 41 we do not think IVIL administered in 3-week intervals leads to accumulation of lipids.

Also, pregnancy per se apparently does not appear to reduce ATAb-activities, because no such suppressive effect was seen over time in the two healthy normal controls during the course of their normal pregnancies. It appears conceivable, that progress of normal pregnancies leads to a normalization of elevated ATAb-activities, while not affecting low or normal ATAb-activities.

Since the mechanism of IVIL on ATAb-activity is unknown, this finding requires further research.

It appears intriguing, that IVIL infusion in several RPL patients apparently produce positive clinical effects even prior to normalizing ATAb-activities. We believe that peripheral blood is not the most relevant compartment for the measurement of ATAb-activities. We suggest that ATAb-activity is probably more relevant close to or in direct contact to the trophoblast, such as in the intervillous space or in the basal plate of the placenta. It appears conceivable, that ATAb-kinetics in these compartments differ from peripheral blood and may get normalized by IVIL infusions much earlier in pregnancy.

Most reports on use of IVIL for implantation and pregnancy success point to effects against a systemic and/or endometrial immunologic over-activation profile, such as Th1-dominance, uNK-cell count and -activation, as well as IFN-γ and TNF−α upregulation..22, 42 We are not aware of studies addressing effects of IVIL infusions on humoral immune responses in general or on anti-trophoblast or anti-enolase antibodies in particular. Our observational study indicates for the first time a significant reduction of elevated ATAb-activity by IVIL, as well as by repetitive infusions during the course of normal pregnancies. Also, the clinical effects in ATAb-positive RPL patients appear promising: All IVIL-treated patients carrying euploid embryos had uneventful pregnancies and delivered healthy term newborns.

These observations await confirmation by adequately powered randomized studies. At this point, this observational study suggests for the first time, that high ATAb-activity in otherwise unexplained RPL patients might be a promising condition to select for effective IVIL-treatment during a consecutive pregnancy.

ACKNOWLEDGMENTS

We thank our patients for participating in this study. Mrs Weimer and Mrs. Herbst helped with their expertise in flow cytometric ATAb-analysis. We thank Dr. Alexander Crispin, LMU-Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie (IBE) for his statistical expertise.

CONFLICT OF INTEREST

Authors disclose no financial and personal conflicts of interest in relation to this study.

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