\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Polyclonal Anti-D Antibodies Significantly Reduce the Rate of
Miscarriages in Rh(D) positive Women with Recurrent Pregnancy loss}



\author[1]{Frauke Ringel}%
\author[1]{Falk Lewandofski}%
\author[1]{Holger Kiesewetter}%
\author[2]{Jalid Sehouli}%
\author[1]{Berthold Hoppe}%
\author[1]{Alina Kiesewetter}%
\author[3]{Reinhard Hannen}%
\author[3]{Christian Friedrich  Stoll}%
\author[4]{Sylvia Maas}%
\author[5]{Gülden Halis}%
\author[2]{Abdulgabar Salama}%
\affil[1]{Medicover, Haemostaseologicum Mitte}%
\affil[2]{Charité Universitätsmedizin Berlin}%
\affil[3]{CERES Kinderwunschzentrum Berlin}%
\affil[4]{Frauenärzte Maas}%
\affil[5]{Kinderwunschteam Berlin}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Objective: Coating of autologous red blood cells (RBCs) with polyclonal
antibodies to Rh(D) antigen may result in an immunomodulation and
improved outcome in Rh(D) positive women with recurrent pregnancy loss
(RPL). Design: Retrospective data analysis. Setting: Rh(D) positive
women with a history of RPL and ineffective treatment with low molecular
weight heparin (LMWH) and/or aspirin Methods: Anti-D (300 \selectlanguage{greek}µ\selectlanguage{english}g) was given
subcutaneously to 60 women either prior to pregnancy and/or two times
within 12 weeks of gestation. Main outcome measures: Births of healthy
child/children. Results: Treatment with Anti-D resulted in successful
pregnancies in 67\% of all cases. The remaining women had only aborts
(23\%) or did not become pregnant (10\%). None of the treated women has
developed anaemia due to this treatment or any other significant adverse
reaction. The rate of successful pregnancies does not appear to be
influenced by the administration of: Anti-D prior to pregnancy, age,
thrombophilia or previous alive births. Conclusion: The improved outcome
following the administration of Anti-D in women with RPL might be
explained by immune modulations induced by different immune reactions
including polarization of decidual macrophages. The results obtained in
this study clearly indicate that Anti-D is safe and highly effective in
treatment of Rh(D) positive women with RPL. However, further studies are
required to support our results and to find out the optimal dose and
timing of Anti-D administration. Funding: There was no funding included.
Key words: abort, Anti-D, aspirin, low molecular weight heparin, new
treatment, recurrent pregnancy loss%
\end{abstract}%



\sloppy


\textbf{Introduction}

Recurrent pregnancy loss (RPL) is defined as two or more consecutive
pregnancy losses prior to 20 -- 24 weeks gestation. It affects about 1
-- 2 \% of pregnancies and is often associated with serious
psychological complications. During the last few decades, many factors
including age, inheritable and aquired genetic and/or anatomical
abnormalities, infections, and endocrine dysfunctions have been
identified to play a crucial role in this field. However, the true cause
remains unclear in most cases. Until now, there is no specific or even
generally accepted treatment in such cases.\textsuperscript{1,2} In
general, pharmacological treatment is only recommended for
anti-phospholipid syndrome, and factor V
mutation.\textsuperscript{3-5}Dependent on clinicians and patients, this
treatment may also be employed in the presence of other congenital
thrombophilias, i.e. prothrombin mutation, deficiencies in
anti-thrombin, protein C, and protein S.\textsuperscript{1,6,7}

Based on the fact that pregnancy reflects an immunological miracle
combining immune defense/response on the one hand and immune tolerance
on the other hand, it is not surprising that immune imbalance during
pregnancy may result in complications. In fact, several studies have
shown that RPL could be related to immune abnormalities in roughly 50 \%
of affected women.\textsuperscript{8-12} Current studies have focused on
the role of decidual macrophages. These cells mature during early stages
of pregnancy and persist during the entire gestation
period.\textsuperscript{13} In the first trimester, about 40 \% of all
cells in the decidua are leukocytes and 20 -- 30 \% of these cells are
macrophages, which have the plasticity to alter their function in
response to various environmental signals. They are also sensitive to
small changes in the microenvironment\textsuperscript{13-15}, they
represent the most crucial immune cells for pregnancies due to their
involvement in regulation of implantation, placentation, fetal
development, and most importantly vascular remodelling at the
maternal-fetal interface. These properties make those cells attractive
as a therapeutic target in immune related diseases.

Polyclonal Anti-D is successfully used to prevent alloimmunization in
Rh(D) negative pregnant women as well as in treatment of patients with
autoimmune thrombocytopenia (ITP). Until now, the mechanisms by which
these antibodies are operating \emph{in vivo} are poorly understood. In
addition to Fc mediated phagocytosis of IgG coated RBCs, Anti-D has
exciting immunomodulatory effects, which could not completely be
explained yet.\textsuperscript{16,17}

In this retrospective study, Anti-D has been shown to increase the rates
of successful pregnancies in Rh(D) positive women who remained abortive
despite treatment with LMWH and/or aspirin.

\textbf{Patients and methods}

Serological testing including blood group, antibody screening, and
direct antiglobulintest were performed by standard techniques using the
gel cards (Biorad, Cressier sur Morat, Switzerland and Grifols SA,
Barcelona, Spain). Only Rh(D) positive women who had at least two
pregnancy losses have been included in this retrospective study. 59
women (age 23 - 45 years) had inheritable genetic abnormalities
(thrombophilias), one women did not give consent to genetic testing but
had thrombophilia history in her anamnesis. All women had ineffective
pretreatment with LMWH and/or aspirin. They were treated on an
outpatient basis between 2016 and 2021 at our practice, the
Haemostaseologicum Mitte in Berlin. All medical records and
investigations were retrospectively reviewed.

Dependend on presentation, unpregnant women were introduced to admit
themselves Anti-D (300 \selectlanguage{greek}µ\selectlanguage{english}g Rhophylac, CSL Behring) five to ten days prior
to the expected ovulation, and two times within the first twelve weeks
of pregnancy. All other women were presented within short time when they
became aware of their pregnancy, and received, similar to the other
group, two times Anti-D.

All women were clinically examined and investigated for underlying
diseases which might be responsible for the RPL. In addition, they were
tested for thrombophilia.

Informed written consent was obtained for participation, and the local
ethics review board was briefed about this study.

Statistical analyses were performed using the IBM SPSS statistics 28.0
software.

\textbf{Results}

As expected, administration of Anti-D resulted in a weakly positive
direct antiglobulin test in all cases, but none of the treated women
developed significant haemolysis.

Most importantly, the majority of these women had not only unremarkable
pregnancy, but also normal delivery. Only six women did not get pregnant
(10 \%), and 40 of the remaining 54 women gave a live birth (60 \% of
all treated women and 74 \% of all pregnant women, respectively) (figure
1).

\textbf{Impact of previous aborts/births on outcome}

Despite treatment with LMWH and/or aspirin, 36 of the 60 women had two
or three aborts prior to Anti-D treatment, and the remaining 24 had four
or more aborts (figure 1). Following treatment with Anti-D, 27 women of
the first group had successful pregnancies (75 \%), nine had aborts (25
\%), and three did not get pregnant (8 \%). Of the second group of 24
women, 13 gave birth (54 \%), nine had aborts (38 \%), and three did not
get pregnant (13 \%).

Prior to treatment with Anti-D, 30 women had no birth, and 30 women had
at least one birth (table 2). Of the first group, 22 women had
successful pregnancies (73 \%), eight had aborts (27 \%), and one women
did not get pregnant (3 \%). Of the other 30 women, 18 women gave birth
(60 \%), ten had aborts (33 \%), and five did not get pregnant (8 \%)
(table 2).

A chi-square test was used to compare number of aborts prior to
treatment with Anti-D and successful pregnancy under treatment with
Anti-D. No expected cell frequencies were below 5, and the results
showed no significant correlation with \selectlanguage{greek}χ\selectlanguage{ngerman}²(1) = 0·303, p(\selectlanguage{greek}χ\selectlanguage{ngerman}²) = 0·582,
p(Fisher's exact test) = 0·784, V = 0·071.

\textbf{Impact of age}

There have been 23 women at ages between 23 and 35 years, and 37 at ages
between 36 to 45 (table 1). Of the first group, 17 women had successful
births (74 \%), six had aborts (26 \%), and two women did not get
pregnant (9 \%). Of the second group, 27 women gave births (73 \%), 11
had aborts (30 \%), and four did not get pregnant (11 \%) (table 2).

A chi-square test was used to compare age groups and successful
pregnancies under treatment with Anti-D. No expected cell frequencies
were below 5, and the results showed no significant correlation with
\selectlanguage{greek}χ\selectlanguage{ngerman}²(1) = 0·881, p(\selectlanguage{greek}χ\selectlanguage{ngerman}²) = 0·348, p(Fisher's exact test) = 0·408, V = 0·121.

\textbf{Impact of Anti-D dose}

Of the 25 women who received Anti-D only after conception, 19 gave birth
(76 \%), and 8 had aborts (32 \%) (table 2). Of the 21 women who
received Anti-D prior to pregnancy, 14 gave birth (67 \%), ten had
aborts (48 \%). Six women did not get pregnant. For eight women full
data of Anti-D administration were not available (table 2).

In summary, the prophylactic administration of Anti-D also prior to
pregnancy does not appear to be more effective than the administration
only post gestation.

A chi-square test was used to compare pre and after conception
injections of Anti-D with successful pregnancies. No expected cell
frequencies were below 5. Results showed no significant correlation with
\selectlanguage{greek}χ\selectlanguage{ngerman}²(1) = 0·801, p(\selectlanguage{greek}χ\selectlanguage{ngerman}²) = 0·371, p(Fisher's exact test) = 0·522, V = 0·132.

\textbf{Impact of blood group}

There have been 31 women with blood group A, from whom 28 got pregnant,
21 gave birth (68 \%), ten had aborts (32 \%). Of the nine women with
blood group B, four gave birth (44 \%), and 5 had aborts (56 \%). Of the
14 women with blood group O, 12 gave birth (86 \%), and four had aborts
(29 \%). Only three women had blood group AB, from whom two gave birth
(67 \%), no woman had an abort (0 \%), and one did not get pregnant
(table 2).

A chi-square test was used to compare blood group and successful
pregnancies. Four cell frequencies were below 5, and the results showed
no significant correlation with \selectlanguage{greek}χ\selectlanguage{ngerman}²(1) = 2·401, p(\selectlanguage{greek}χ\selectlanguage{ngerman}²) = 0·493, p(Fisher's
exact test) = 0·518, V = 0·205.

\textbf{Impact of thrombophilia associated mutations}

All women showed thrombophilia associated mutations, except one who did
not give consent for this kind of genetic testing. 41 women had a PAI1
mutation, 39 a prothrombin 19911 mutation, 27 a fibrinogen alpha
mutation, 19 a fibrinogen gamma mutation, seven a factor V Leiden
mutation, six a HR2 mutation, two a prothrombin 20210 mutation, two a
FSAP mutation, one a MTHFR mutation, and none had a tPA mutation.

From 16 women with only one mutation 13 gave births (81 \%), two had
aborts (13 \%), and one did not get pregnant (6 \%). From 16 women with
two mutations 12 gave births (75 \%), three had aborts (19 \%), and one
did not get pregnant (6 \%). Three mutations were found with 15 women,
of whom seven gave births (47 \%), seven had aborts (47 \%), and three
did not get pregnant (20 \%). Four to six mutations were found in 12
women. Of these women, seven gave births (64 \%), five had aborts (45
\%), and one woman did not get pregnant (9 \%) (table 2).

A chi-square test was used to compare number of thrombophilia associated
mutations (one or two versus three or more) and successful pregnancies.
No expected cell frequencies were below 5. Results showed no significant
correlation with \selectlanguage{greek}χ\selectlanguage{ngerman}²(1) = 1·701, p(\selectlanguage{greek}χ\selectlanguage{ngerman}²) = 0·192, p(Fisher's exact test) =
0·276, V = 0·170.

\textbf{Discussion}

\textbf{Main findings}

The vast majority of women treated with Anti-D had unremarkable
pregnancy and delivery. More than 70\% of all pregnant women delivered
healthy children. Interestingly, the efficacy of this treatment does not
appear to be dependent on age, thrombophilia, and prior treatment.
Nevertheless, by these data there could be a relation between those
parameters and outcome of a pregnancy, but case numbers are too low for
significant statistical analysis. Treatment with Anti-D was safe with no
signs of significant hemolysis.

Worldwide, RPL represents a serious and unresolved
problem.\textsuperscript{1,2,6} Immune pathways play a key role in the
pathophysiology of RPL. Already at early stage, gestation induces a
major immune response which presumably involves all immune cells,
including lymphocytes, macrophages and decidual dendritic cells. Thus,
the question whether immunotherapy may help in management of RPL is
increasingly focused in new studies.\textsuperscript{18}

Macrophages are presumably the main player in regulation of pregnancy,
including implantation, placentation, fetal development, parturition,
and most importantly vascular remodeling at the maternal-fetal
interface. Thus, some modulation of macrophages may prevent miscarriages
related to immune imbalance. On this, the question rised, whether a
subtle attraction of these cells by coating of autologous red blood
cells in such affected women may result in successful pregnancies. In
fact, the results obtained during observation were encouraging. The vast
majority of treated women had unremarkable pregnancy and delivery.
Interestingly, the efficacy of this treatment does not appear to be
dependent on age, thrombophilia, and prior treatment. Nevertheless, by
these data there could be a relation between those parameters and
outcome of a pregnancy, but case numbers are too low for significant
statistical analysis. Since all treated women received simultaneously
LMWH and/or aspirin, the question remains open whether the beneficial
effect was solely related to Anti-D or to a combined effect of both
drugs. This is supported by the finding that LWMH beyond its
anti-coagulating effect may induce
immunomodulation.\textsuperscript{19}Until now, a monotherapy with
Anti-D has not been undertaken. However, it has been shown that
treatment with intravenous immunoglobulins (IvIg) that somewhat
resembles Anti-D, may improve the outcome in pregnant women with
miscarriages.

Similarly, there is evidence that polyclonal Anti-D has an
immunomodulatory effect, which could not fully be explained
yet.\textsuperscript{20,21} In our experience, this treatment is safe
when it is given subcutaneously or at least slowly by intravenous
route.\textsuperscript{22} In previous studies we have demonstrated that
the administration of LMWH and/or aspirin results in live births in most
women with RPL.\textsuperscript{7} In this retrospective study, we used
low Anti-D doses (2 to 3 times 300 \selectlanguage{greek}µ\selectlanguage{english}g) for treatment of RPL in Rh(D)
positive women who remained abortive despite treatment with LMWH and/or
aspirin.

At first glance, the question may arise whether this treatment could be
justified in Rhesus positive pregnant women. Based on the following
facts, this treatment is, we think, rational as long as no alternative
therapy is available for such affected patients. Anti-D is used in
treatment of adults and children since four decades with little or no
side effects, if the drug was carefully injected.\textsuperscript{22}The
injection of 300 \selectlanguage{greek}µ\selectlanguage{english}g is harmless compared to that dose given in ITP (50 -
75 \selectlanguage{greek}µ\selectlanguage{english}g/kg body weight), and to that amount reaching Rh(D) positive
infants in Rh(D) negative pregnant women who receive at least two times
100 - 300 \selectlanguage{greek}µ\selectlanguage{english}g prophylaxis.

The positive influence of Anti-D on RPL is evident as reflected by the
significantly increased rates of successful pregnancies in the majority
of treated women in our practice. This effect does not appear to be
dependent on age, inherited haemophilias or blood group. The question
whether Anti-D per se or only in combination with LMWH is effective is a
matter of speculation. Since the mechanisms by which LMWH influences the
outcome in RPL is largely unknown, a synergic effect can not be
excluded. Ultimately, LMWH has been shown to induce \emph{in vitro}
and\emph{in vivo} a proinflammatory profile on RPL. It has been
suggested that this effect could be valuable at the implantation
stage.\textsuperscript{7} Thus, it remains unclear whether Anti-D alone
could also be effective as well as the combination with LMWH.

The question why this treatment was ineffective in 26 \% of the treated
pregnant women remains to be answered. A possible explanation could be
related to unknown predisposition factors that cannot be influenced by
immunological modulations. Similarly, the phenomenon that the treatment
may again result in miscarriage in some women who had been shown to be
responsive remains also obscure. Whether this ineffectivity could be
related to the giving dose or timing of Anti D admininistration remains
to be answered.

Though, the most possible explanation for the successful pregnancy
following treatment with Anti-D may be attributed to an immunomodulatory
effect, some other questions remain open. For example, when and by which
mechanisms Anti-D may induce the tolerance of the semiallogeneic fetus.
There is no doubt that pregnancy is surroundingly controlled by immune
reactions which simultaneously maintain immune response (defense) and
tolerance (semi-allogeneicity), respectively. \textsuperscript{10,11,23}

Changes during pregnancy include an increase in phagocytic cells and
their functional capacity to ingest IgG coated
RBCs,\textsuperscript{24}an increase of T and B regulatory cells,
reduction of natural killer cells, and polarization of decidual
macrophages.\textsuperscript{23,25-28} It has been demonstrated that RPL
is associated with an imbalance of pregnancy related immune
haemostasis,\textsuperscript{10,11,29} which could, at least in part, be
normalized by immunomodulation.\textsuperscript{18,30}

Anti-D administration into Rh(D) positive pregnant women results in
coating of autologous RBCs with IgG antibodies. This happening
represents a dangerous signal, and attracts macrophages to recognize IgG
opsonized RBCs. Although the attracted macrophages do not appear to
ingest a significant amount of the IgG coated RBCs, they could be
influenced in the presence of IgG-coated RBCs.

In addition, polyclonal Anti-D may contain antibodies other than Anti-D
which may have influence on macrophages, independent of phagocytosis,
i.e. HLA and anti-idiotype antibodies. Thus, it would not be surprising
that the reactions to Anti-D may lead to polarization of decidual
macrophages. These cells exhibit wide plasticity and are sensitive to
small microenvironmental changes, including nutrition, smoking, toxins,
inflammation, and physiological stress.\textsuperscript{13-15}

It seems that the M1 subtype predominates over the M2 subtype in RPL.
Coating of autologous RBCs with antibodies represents unphysiological
condition \emph{in vivo} , which may lead to polarization of M1 to M2
phenotype. The latter cells seem to be responsible for immunotolerance,
which is a prequisite for successful implantation a fetal development.

This hypothesis can only be proved by phenotyping of decidual
macrophages prior to and following treatment. Alternatively, an extended
investigation of cytokines and changes of peripheral immune cells may
give an explanation of this phenomenon.\textsuperscript{8-11,26,28}

\textbf{Strength and limitations}

We are aware of the limitation of the presented study. It is mainly
based on retrospective data. In addition, the number of treated women is
relatively small and too low for significant statistical analysis, and
an optimal dose and timing of Anti-D remained speculative.

\textbf{Conclusions}

Anti-D could be a new therapeutic option in women with recurrent
pregnancy loss who are not responding well to low molecular heparin
and/or aspirin alone.

Thus, new well designed studies may gain deeper insights in this field,
and into the maternal-fetal immune interface.

\textbf{Statement of Ethics}

This is a retrospective analysis of patient data. The Ethics Committee
of the Berlin Medical Association has confirmed that no ethical approval
is required.

\textbf{Declaration of interests}

The authors have no relevant financial or non-financial interests to
disclose.

\textbf{Funding Sources}

The authors declare that no funds, grants, or other support were
received during the preparation of this manuscript.

\textbf{Author Contribution statement}

FR: literature search, data collection, data analysis, data
interpretation, writing, critical revision, accessed and verified all
data; FL: data collection; HK: treatment of patient, critical revision;
JS: writing, critical revision; BH: treatment of patients, critical
revision; AK: data collection; RH: treatment of patient, critical
revision; CFS: treatment of patient, critical revision, SM: treatment of
patient, critical revision; GH: treatment of patient, critical revision;
AS: treatment of patient, literature search, data interpretation,
writing, critical revision, accessed and verified all data

All authors confirm that they had full access to all the data in the
study and accept responsibility to submit for publication of the
manuscript.

\par\null

\textbf{References}

1. Hong Li Y, Marren A. Recurrent pregnancy loss: A summary of
international evidence-based guidelines and practice. \emph{Aust J Gen
Pract} 2018; \textbf{47} (7): 432-6.

2. Green DM, O'Donoghue K. A review of reproductive outcomes of women
with two consecutive miscarriages and no living child. \emph{J Obstet
Gynaecol} 2019; \textbf{39} (6): 816-21.

3. Practice Committee of the American Society for Reproductive M.
Evaluation and treatment of recurrent pregnancy loss: a committee
opinion. \emph{Fertil Steril} 2012; \textbf{98} (5): 1103-11.

4. Homer HA. Modern management of recurrent miscarriage. \emph{Aust N Z
J Obstet Gynaecol} 2019; \textbf{59} (1): 36-44.

5. Henkel A, Shaw KA. Advances in the management of early pregnancy
loss. \emph{Curr Opin Obstet Gynecol} 2018; \textbf{30} (6): 419-24.

6. Rai R, Regan L. Recurrent miscarriage. \emph{Lancet}
2006;\textbf{368} (9535): 601-11.

7. Monien S, Kadecki O, Baumgarten S, Salama A, Dorner T, Kiesewetter H.
Use of heparin in women with early and late miscarriages with and
without thrombophilia. \emph{Clin Appl Thromb Hemost} 2009;\textbf{15}
(6): 636-44.

8. Luan X, Kang X, Li W, Dong Q. An investigation of the relationship
between recurrent spontaneous abortion and memory T follicular helper
cells. \emph{Am J Reprod Immunol} 2017; \textbf{78} (5).

9. Qian J, Zhang N, Lin J, et al. Distinct pattern of Th17/Treg cells in
pregnant women with a history of unexplained recurrent spontaneous
abortion. \emph{Biosci Trends} 2018; \textbf{12} (2): 157-67.

10. Yang F, Zheng Q, Jin L. Dynamic Function and Composition Changes of
Immune Cells During Normal and Pathological Pregnancy at the
Maternal-Fetal Interface. \emph{Front Immunol} 2019; \textbf{10} : 2317.

11. Deshmukh H, Way SS. Immunological Basis for Recurrent Fetal Loss and
Pregnancy Complications. \emph{Annu Rev Pathol} 2019; \textbf{14} :
185-210.

12. Saini V, Arora S, Yadav A, Bhattacharjee J. Cytokines in recurrent
pregnancy loss. \emph{Clin Chim Acta} 2011; \textbf{412} (9-10): 702-8.

13. Jena MK, Nayak N, Chen K, Nayak NR. Role of Macrophages in Pregnancy
and Related Complications. \emph{Arch Immunol Ther Exp (Warsz)}
2019;\textbf{67} (5): 295-309.

14. Zhang YH, He M, Wang Y, Liao AH. Modulators of the Balance between
M1 and M2 Macrophages during Pregnancy. \emph{Front Immunol}
2017;\textbf{8} : 120.

15. Yao Y, Xu XH, Jin L. Macrophage Polarization in Physiological and
Pathological Pregnancy. \emph{Front Immunol} 2019; \textbf{10} : 792.

16. Lazarus AH, Crow AR. Mechanism of action of IVIG and anti-D in
ITP.\emph{Transfus Apher Sci} 2003; \textbf{28} (3): 249-55.

17. Brinc D, Denomme GA, Lazarus AH. Mechanisms of anti-D action in the
prevention of hemolytic disease of the fetus and newborn: what can we
learn from rodent models? \emph{Curr Opin Hematol} 2009; \textbf{16}
(6): 488-96.

18. Carp H. Immunotherapy for recurrent pregnancy loss. \emph{Best Pract
Res Clin Obstet Gynaecol} 2019; \textbf{60} : 77-86.

19. Rasmark Roepke E, Bruno V, Nedstrand E, et al.
Low-molecular-weight-heparin increases Th1- and Th17-associated
chemokine levels during pregnancy in women with unexplained recurrent
pregnancy loss: a randomised controlled trial. \emph{Sci Rep}
2019;\textbf{9} (1): 12314.

20. Coopamah MD, Freedman J, Semple JW. Anti-D initially stimulates an
Fc-dependent leukocyte oxidative burst and subsequently suppresses
erythrophagocytosis via interleukin-1 receptor antagonist.
\emph{Blood}2003; \textbf{102} (8): 2862-7.

21. Crow AR, Lazarus AH. The mechanisms of action of intravenous
immunoglobulin and polyclonal anti-d immunoglobulin in the amelioration
of immune thrombocytopenic purpura: what do we really
know?\emph{Transfus Med Rev} 2008; \textbf{22} (2): 103-16.

22. Salama A. Emerging drugs for immune thrombocytopenia
(ITP).\emph{Expert Opin Emerg Drugs} 2017; \textbf{22} (1): 27-38.

23. Schumacher A, Sharkey DJ, Robertson SA, Zenclussen AC. Immune Cells
at the Fetomaternal Interface: How the Microenvironment Modulates Immune
Cells To Foster Fetal Development. \emph{J Immunol} 2018;\textbf{201}
(2): 325-34.

24. Davis D, Kaufmann R, Moticka EJ. Nonspecific immunity in pregnancy:
monocyte surface Fcgamma receptor expression and function. \emph{J
Reprod Immunol} 1998; \textbf{40} (2): 119-28.

25. Huang N, Chi H, Qiao J. Role of Regulatory T Cells in Regulating
Fetal-Maternal Immune Tolerance in Healthy Pregnancies and Reproductive
Diseases. \emph{Front Immunol} 2020; \textbf{11} : 1023.

26. Pei CZ, Kim YJ, Baek KH. Pathogenetic factors involved in recurrent
pregnancy loss from multiple aspects. \emph{Obstet Gynecol Sci}
2019;\textbf{62} (4): 212-23.

27. Robertson SA, Care AS, Moldenhauer LM. Regulatory T cells in embryo
implantation and the immune response to pregnancy. \emph{J Clin
Invest}2018; \textbf{128} (10): 4224-35.

28. Esteve-Sole A, Luo Y, Vlagea A, et al. B Regulatory Cells: Players
in Pregnancy and Early Life. \emph{Int J Mol Sci} 2018; \textbf{19} (7).

29. Matthiesen L, Kalkunte S, Sharma S. Multiple pregnancy failures: an
immunological paradigm. \emph{Am J Reprod Immunol} 2012; \textbf{67}
(4): 334-40.

30. Muyayalo KP, Li ZH, Mor G, Liao AH. Modulatory effect of intravenous
immunoglobulin on Th17/Treg cell balance in women with unexplained
recurrent spontaneous abortion. \emph{Am J Reprod Immunol}
2018;\textbf{80} (4): e13018.

\textbf{Hosted file}

\verb`Figure 1 BJOG.docx`  available at \url{https://authorea.com/users/557625/articles/607212-polyclonal-anti-d-antibodies-significantly-reduce-the-rate-of-miscarriages-in-rh-d-positive-women-with-recurrent-pregnancy-loss}

\textbf{Hosted file}

\verb`Tables BJOG.docx`  available at \url{https://authorea.com/users/557625/articles/607212-polyclonal-anti-d-antibodies-significantly-reduce-the-rate-of-miscarriages-in-rh-d-positive-women-with-recurrent-pregnancy-loss}

\selectlanguage{english}
\FloatBarrier
\end{document}

