Journal of Reproductive Immunology 150 (2022) 103476

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Journal of Reproductive Immunology
journal homepage: www.elsevier.com/locate/jri

The role of epidermal growth factor-like domain-related abnormalities,
protein S Tokushima, and anti-protein S autoantibodies in pregnancy loss
Yoshihiro Sato a, b, Toshitaka Sugi a, *, Rie Sakai a, Hiroyuki Kuma caLaboratory for Recurrent Pregnancy Loss, Sugi Women’s Clinic, Kanagawa, Japan
Yoshihiro Women’s Clinic, Tokyo, Japanc
Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Protein S
Protein S Tokushima
Autoantibodies
Pregnancy loss
Epidermal growth factor

Background: Protein S (PS) deficiency and autoantibodies that bind to PS (anti-PS) have been described in pa­
tients with adverse pregnancy outcomes, including pregnancy loss. PS Tokushima is a congenital abnormality ofthe second epidermal growth factor (EGF)-like domain, and anti-PS has been reported to recognize EGF-likedomains.
Objectives: We evaluated the role of PS Tokushima and anti-PS in patients with pregnancy loss.
Methods: Patients with recurrent early pregnancy loss (n = 324; group A), those with one or more mid-to-latepregnancy loss (n = 196; group B), and infertile women having no pregnancy loss (n = 650; group C) werescreened for PS type II deficiency and anti-PS. Patients who were diagnosed with PS type II deficiency underwent
genetic analysis for the detection of PS Tokushima.
Results: The incidence of patients with PS Tokushima was 1.85 %, 5.10 %, and 1.23 % in groups A, B, and C,
respectively. The incidence of patients with PS Tokushima was significantly higher in group B (p = 0.0027) thanin group C. The incidence of patients with anti-PS was 20.1 %, 23.0 %, and 19.2 % in groups A, B, and C,
respectively. The incidence of patients with anti-PS was significantly higher in groups A (p = 0.0229), B (p =0.0071), and C (p = 0.0288) than in previously reported healthy nonpregnant women (7.1 %, 4/56).
Conclusions: Our data suggest that PS Tokushima is associated with mid-to-late pregnancy loss, while anti-PS areassociated with recurrent early pregnancy loss, mid-to-late pregnancy loss, and infertility.

1. Introduction
Protein S (PS) is a vitamin K-dependent glycoprotein that functions
as a cofactor to activated protein C (APC), which is an anticoagulant
serine protease, and is an important regulator of blood coagulation(Rezende et al., 2004). Mature PS has a modular structure consisting of aγ-carboxyglutamic acid domain, a thrombin-sensitive region, four
epidermal growth factor (EGF)-like domains (EGF1-4), and a sex
hormone-binding globulin-like domain containing two laminin G-like-­
domain-like repeats.
Recently, many studies have suggested an association between
adverse pregnancy outcomes and PS deficiency (Rey et al., 2003; Rob­
ertson et al., 2006; Paidas et al., 2005; Hojo et al., 2008; Ebina et al.,2015). Rey et al. performed a meta-analysis and reported that PS defi­
ciency was associated with recurrent pregnancy loss and fetal loss after

22 weeks (Rey et al., 2003). Robertson et al. reported in a systematic
review that PS deficiency was associated with late fetal loss (Robertson
et al., 2006).
PS Tokushima (p.Lys196Glu), a genetic mutation and one of themolecular defects identified in EGF2 of PS (Yamazaki et al., 1993a;
Hayashi et al., 1994), is found in 1.6 %–1.8 % of healthy Japanese in­dividuals, whereas it is found in 5 %–10 % of Japanese patients withvenous thromboembolism (VTE) (Kinoshita et al., 2005; Miyata et al.,2009; Yamazaki et al., 1993b; Kimura et al., 2006). This mutation was
responsible for PS type II deficiency, namely, PS qualitative deficiency,
which is characterized by decreased PS activity with normal PS antigen
levels (Hayashi et al., 1994; Tsuda et al., 2002; Gandrille et al., 2000).
Some studies have described autoantibodies that bind to PS (anti-PS)
in patients with adverse pregnancy outcomes (Marozio et al., 2011; Satoet al., 2018). It was reported that 20 out of 100 patients with recurrent* Corresponding author at: Laboratory for Recurrent Pregnancy Loss, Sugi Women’s Clinic, 2-12-1-7F, Shinyokohama, Kohoku, Yokohama, Kanagawa, 222-0033,
Japan.
E-mail address: sugi@sugi-wc.jp (T. Sugi).
https://doi.org/10.1016/j.jri.2022.103476
Received 19 November 2021; Received in revised form 1 January 2022; Accepted 9 January 2022
Available online 11 January 2022
0165-0378/© 2022 Elsevier B.V. All rights reserved.

Y. Sato et al.

Journal of Reproductive Immunology 150 (2022) 103476

pregnancy loss were seropositive for anti-PS, which recognized EGF1-4
in PS (Sato et al., 2018).
PS Tokushima and anti-PS are common in the EGF-like domainrelated abnormalities in PS. In this study, we investigated the association
among PS Tokushima, anti-PS, and recurrent early and mid-to-latepregnancy loss.
2. Materials and methods

the exon and exon-intron boundary regions were amplified using poly­merase chain reaction (PCR). PS Tokushima samples were analyzed by
PCR-restriction fragment length polymorphism with a mutagenic primer(5′ − CCATCCTGCTCTTACCTTTACAATCTGACT-3′ ) and a normal primer(5′ -CTCCTGAAAAGTTCTCTGCA-3′ ). The amplified fragment from themutant allele was digested by Hinf I, and two fragments (404 and 30 bp)
were detected. In contrast, the normal allele yielded only a 434-bp
fragment after enzyme digestion (Yamazaki et al., 1993b).

2.1. Patients

2.4. SDS-PAGE and immunoblotting for Anti-PS

From May 2018 to February 2020, plasmas from 324 patients withrecurrent early pregnancy loss (group A), 196 patients with one or more
mid-to-late pregnancy loss (group B) and 650 infertile women having no
pregnancy loss (group C) who were referred to Sugi Women’s Clinic
were screened for PS-specific activity and anti-PS by immunoblotting.
Patients who were diagnosed with PS type II deficiency by low PSspecific activity underwent genetic analysis for the detection of PS
Tokushima.
Patients with recurrent early pregnancy loss (group A) had three ormore pregnancy losses before 10 weeks gestation. Those with mid-tolate pregnancy loss (group B) had one or more pregnancy losses after
10 weeks gestation. Infertile women (group C) had no pregnancy loss.
They were patients with repeated implantation failure defined as having
three or more embryo transfers with good-quality embryos. We did notenroll patients with repeated implantation failure into the group A nor
the group B. Ectopic pregnancy and/or elective abortion were excluded.
The main biological and clinical characteristics of the three groups ofpatients are given in Table 1. No patient was pregnant and taken any
treatment when plasma was obtained.
Blood samples were collected in non-activating plastic tubes con­taining 3.2 % trisodium citrate (9:1 v/v). After centrifugation, plasma
aliquots were immediately stored at − 60 ◦ C until use. Informed consent
was obtained from all subjects. The study protocol was approved by the
local ethics review committee.

SDS-PAGE was performed using 10 % polyacrylamide gel. PS(Enzyme Research Laboratories, IN, USA) was prepared in non-reducing
forms. PS (5 μL of 10 μg/mL solution) was applied to each lane. Transfer
to polyvinylidene difluoride (PVDF) membrane was done for 20 min at
0.1 amps. Membranes were blocked for 1.5 h with 1% bovine serum
albumin (BSA) in Tris-buffered saline (TBS) at pH 7.3. Incubation withpatient plasma (1/100) was done for 2 h followed by three washes with
0.05 % Tween 20/TBS. The membrane was exposed to horseradish
peroxidase-conjugated polyclonal antibodies to human IgG or IgM for 1
h followed by washing as above. Immunobands were developed using
3,3′ ,5,5′ -tetramethylbenzidine.
In our immunoblot, we evaluated the intensity of immunobands
visually compared with negative controls. A plasma was considered
positive if anti-PS IgG and/or IgM was positive.
2.5. Statistical analysis
Differences between the two groups were analyzed for statisticalsignificance (P < 0.05) by the Fisher’s exact test.
3. Results
As shown in Table 2, the incidence of patients who were diagnosed
with PS type II deficiency was 2.47 % (8/324), 5.61 % (11/196), and1.54 % (10/650) in groups A, B, and C, respectively. The incidence of
patients with PS type II deficiency was significantly higher in patientswith one or more mid-to-late pregnancy loss (p = 0.0030, odds ratios[OR]: 3.81, 95 % confidence interval [CI]: 1.59–9.10) than in infertile
women having no pregnancy loss.
The incidence of patients with PS Tokushima was 1.85 % (6/324),
5.10 % (10/196), and 1.23 % (8/650) in groups A, B, and C, respec­tively. The incidence of patients with PS Tokushima was significantly
higher in patients with one or more mid-to-late pregnancy loss (p =0.0027, OR: 4.31, 95 % CI: 1.68–11.09) than in infertile women having
no pregnancy loss.
As shown in Table 2, the incidence of patients with anti-PS was 20.1
% (65/324), 23.0 % (45/196), and 19.2 % (125/650) in groups A, B, andC, respectively. The incidence of patients with anti-PS was significantly
higher in groups A (p = 0.0229, OR: 3.26, 95 % CI: 1.14–9.35), B (p =0.0071, OR: 3.87, 95 % CI: 1.33–11.30), and C (p = 0.0288, OR: 3.10, 95% CI: 1.10–8.72) than in our previously reported healthy nonpregnant

2.2. Protein S-specific activity
The total PS activity and antigen levels were measured by thechromogenic method and latex agglutination method, respectively,
using commercial kits (Shino-Test, Tokyo, Japan). The total PS activity
to total PS antigen level ratio was defined as PS-specific activity. Basedon data on healthy individuals from a previous study, the cutoff value todiagnose PS type II deficiency by PS-specific activity was 0.78 (mean－
3.0 standard deviation) (Tsuda et al., 2012; Noguchi et al., 2019).
2.3. Gene analyses
Genomic DNA was prepared from peripheral blood leukocytes, andTable 1
Characteristics of the three groups of women in this study.

No. of patients
Age, mean (range)
No. of pregnancy
losses, mean
(range)Before 10th
week ofpregnancy
Beyond 10th
week ofpregnancy

Recurrent earlypregnancy loss

Mid-to-latepregnancy loss

(Group A)(Group B)

Infertile women
having no
pregnancy loss
(Group C)

324
36.7 (25–47)

196
34.4 (24–46)

650
38.5 (25–53)

3.3 (3–11)

1.5 (1–5)

00

1.1 (1–4)

0

Table 2
Positive rates for PS type II deficiency, PS Tokushima, and anti-PS autoanti­
bodies (anti-PS).

PS type II
deficiency
PS Tokushima
Anti-PS

PS; protein S.
2

Recurrent earlypregnancy loss

Mid-to-latepregnancy loss

Infertile women
having no pregnancy
loss(n = 650; group C)(n = 324; group A)(n = 196; groupB)

8/324 (2.47 %)11/196 (5.61 %)10/650 (1.54 %)6/324 (1.85 %)
65/324(20.1 %)10/196 (5.10 %)45/196(23.0 %)8/650 (1.23 %)125/650(19.2 %)Y. Sato et al.

Journal of Reproductive Immunology 150 (2022) 103476

women (7.1 %, 4/56) (Sato et al., 2018). Anti-PS were associated withrecurrent early pregnancy loss, mid-to-late pregnancy loss, andinfertility.
Table 3 shows the positivity rates for anti-PS in patients with orwithout PS Tokushima. In group A, the incidence of anti-PS was 0.0 %(0/6) and 20.4 % (65/318) in PS Tokushima and non-PS Tokushima,respectively. In group B, the incidence of anti-PS was 10.0 % (1/10) and23.7 % (44/186) in PS Tokushima and non-PS Tokushima, respectively.
In group C, the incidence of anti-PS was 25.0 % (2/8) and 19.2 % (123/642) in PS Tokushima and non-PS Tokushima, respectively. In allgroups, there was no association between anti-PS and PS Tokushima(group A: p = 0.6040, group B: p = 0.4593, group C: p = 0.6539).

Table 3
Positive rates for anti-PS autoantibodies (anti-PS) in patients with or without PS
Tokushima.

PS Tokushima(+)
PS Tokushima(− )Recurrent earlypregnancy loss

Mid-to-latepregnancy loss

Infertile women
having no pregnancy
loss(n = 650; group C)(n = 324; group A)(n = 196; groupB)

0/6 (0.0 %)1/10 (10.0 %)

2/8 (25.0 %)65/318 (20.4 %)44/186 (23.7 %)123/642 (19.2 %)PS; protein S.

4. Discussion

negative influence of thrombophilia is unclear at the stage of implan­tation. In this study, PS Tokushima, a type of thrombophilia, was not
associated with infertility.
EGF family proteins play an important role in implantation. In
particular, heparin-binding EGF-like growth factor (HB-EGF) is crucial
for normal implantation and is involved in blastocyst adhesion anddevelopment (Raab et al., 1996; Leach et al., 2004).
From these findings, it is reasonable to believe that the recognition of
EGF-like domains in PS and EGF family proteins by anti-PS is associatedwith repeated implantation failure.
This study had two limitations. First, only PS type II-deficient pa­
tients that were identified by a low PS-specific activity were tested forgenetic analysis of PS Tokushima. However, it was reported that the PSspecific activity had a sensitivity of 100.0 % for diagnosing PS
Tokushima (Noguchi et al., 2019); therefore, it is highly probable thatpatients who were negative for PS type II deficiency did not have PS
Tokushima. Second, there were no healthy controls in this study. Further
studies performing the genetic analysis of PS Tokushima for all patientsand screening healthy controls are necessary to obtain more accurate
results.
In summary, our data suggested that PS Tokushima is associated with
mid-to-late pregnancy loss, while anti-PS is associated with recurrent
early pregnancy loss, mid-to-late pregnancy loss, and repeated implan­tation failure. Although PS Tokushima and anti-PS are common in the
EGF-like domain-related abnormaities in PS, no direct association wasestablished. PS Tokushima and anti-PS may have different
pathogenecities.

To the best of our knowledge, this is the first study to suggest an
association between PS Tokushima and mid-to-late pregnancy loss andbetween anti-PS and repeated implantation failure as well as pregnancy
loss.
In a previous report, genetic analysis of 304 healthy subjects (168
men aged 22–74 years and 136 women aged 23–74 years) revealed PS
Tokushima in 5 subjects (1.6 %, 5/304) (Kinoshita et al., 2005). In thisstudy, PS Tokushima was detected in 5.10 % of the patients with
mid-to-late pregnancy loss, which is up to 4-fold higher than that
detected in infertile women having no pregnancy loss (p = 0.0027, OR:
4.31, 95 % CI: 1.68–11.09) and up to 3-fold higher than that detected inpreviously reported healthy controls (p = 0.0329, OR: 3.22, 95 % CI:
1.08–9.55) (Kinoshita et al., 2005).
The results of our study are consistent with the results of previous
reports. A meta-analysis showed an association between PS deficiency
and nonrecurrent fetal loss after 22 weeks of gestation (OR: 7.39, 95 %CI: 1.28–42.63) (Rey et al., 2003). A systematic review showed that PS
deficiency was significantly associated with late pregnancy loss (OR:20.09; 95 % CI: 3.70–109.15) (Robertson et al., 2006).
In this study, no association was observed between PS Tokushima
and recurrent early pregnancy loss. This result is also consistent with aprevious report in which 335 patients with recurrent early pregnancy
loss were investigated (Matsukawa et al., 2017).
The frequency of anti-PS was significantly higher in patients withrecurrent early pregnancy loss, mid-to-late pregnancy loss, and infer­tility than in previously reported healthy controls (Sato et al., 2018).
There was no association between PS Tokushima and anti-PS, suggestingthat these may have different pathogenicities.
PS Tokushima was responsible for PS type II deficiency, which causes
thrombophilia (Hayashi et al., 1994; Tsuda et al., 2002; Gandrille et al.,2000), although the inhibition of PS activity by anti-PS depended on theantigenic binding sites of anti-PS. The recognition of EGF1-2 by anti-PS
may inhibit PS activity, causing thrombophilia and leading to pregnancy
loss, similar to PS Tokushima (Sato et al., 2018).
An interesting hypothesis of the pathogenicity of anti-PS is that therecognition of EGF-like domains in PS and EGF family proteins by antiPS may inhibit uterine and placental angiogenesis that is crucial for thesurvival of the embryo and fetus, leading to pregnancy loss (Sato et al.,2018). PS has been known as a natural anticoagulant protein, and it has
been suggested to have an important role in multiple biological pro­
cesses, including coagulation, apoptosis, angiogenesis/vasculogenesis,
and cancer progression (Suleiman et al., 2013). PS was observed toaccumulate around damaged placental trophoblastic cells in early andlate pregnancy, which indicated a possibility that PS could protect orrestore damaged villi and had physiological effects on the placenta(Matsumoto et al., 2008).
Autoantibodies, which recognize EGF-like domains in PS, might berelated to the inhibition of angiogenesis and clearance of apoptotic cells
during normal pregnancy (Sato et al., 2018).
In this study, it was found that anti-PS were associated with not only
pregnancy loss but also repeated implantation failure. However, theAddendumStudy concept and design, Y. Sato, T. Sugi, and H. Kuma; acquisitionof data, Y. Sato, T. Sugi, and R. Sakai; analysis and interpretation of data,
Y. Sato, T. Sugi, and H. Kuma; patient inclusion and management, Y.
Sato and T. Sugi; drafting of the manuscript, Y. Sato, T. Sugi, and H.
Kuma; study supervisor, T. Sugi.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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