Activated protein C resistance impact on Syrian candidates for in vitro fertilisation and the benefit of anticoagulation therapy: a retrospective cohort study

Abstract Activated protein C resistance (APCR) is a common thrombophilia, caused mainly by a mutation. The impact of APCR on the efficacy of In Vitro Fertilization (IVF) are still unclear, and no solid recommendations for its management were published. To investigate the effect of APCR on IVF outcomes and assess the efficacy of our management protocol, we retrospectively scanned the medical records of women who were tested with APCR assay in 2019 at our fertility centre. The 66 women (12%) positive for APCR had lower odds of reaching clinical pregnancies after IVF 0.18 [95% CI: 0.07–0.47] and fewer live births. The administration of low-molecular-weight heparin and aspirin associated with more implantation in treated compared to untreated APCR-positive women with an odds ratio of 43.2 [7.51–248.6]. In conclusion, APCR negatively affects the number of clinical pregnancies after IVF, but anticoagulation therapy can mitigate this effect and significantly increase clinical pregnancies. Impact Statement What is already known on this subject? The evidence about the impact of APCR on IVF outcomes is still inconclusive. According to the Canadian guideline, routine screening for thrombophilia in patients with recurrent pregnancy loss is not recommended. No clear recommendations regarding the management of APCR in the planning for IVF are yet available. What do the results of this study add? APCR significantly increases implantation failure among infertile women who conduct IVF. Management of APCR using LMWH and aspirin was effective in mitigating this effect and increasing successful implantation. What are the implications of these findings for clinical practice and/or further research? Our findings can support the recommendation to include APCR assay in the routine tests for infertile women conducting IVF, and suggest the combination between LMWH and aspirin as an effective therapy to increase successful implantation in APCR positive candidates. However, more controlled clinical trials are still needed to confirm our results.


Introduction
Assisted reproductive technologies (ARTs) are novel techniques that entered the clinical practice in 1978 when the first tube-baby was born (Yovich 2020). In vitro fertilisation (IVF) is the most common and effective type of ARTs. It starts with fertilising the oocytes outside the body, providing an incubating environment for the embryos to evolve, and then transferring them into the uterus (CDC 2019; MedlinePlus 2021). Despite the advanced technology used, the success rates of IVF remain low, and many women are still unable to reach clinical pregnancy (Bahadur et al. 2020). This may be attributed to various reasons, including female age, which increases chromosomal abnormalities (i.e. aneuploidy; Kuliev et al. 2003;Dang et al. 2019), low oocyte quality (Navot et al. 1991), increased body mass index (Rittenberg et al. 2011), and hydrosalpinx (Zeyneloglu et al. 1998). Besides, controversy about the contribution of causes like thrombophilia continues to exist (Qublan et al. 2006;Tan et al. 2016).
Activated protein C resistance (APCR) is a common thrombophilia, with a worldwide prevalence of 10%-15% (Winkler 2019). It causes a thrombophilic state due to the resistance against activated protein C anticoagulant properties. People with this condition lack the normal function of activated protein C pathway (Nicolaes and Dahlb€ ack 2003). APCR causes are classified into inherited and acquired, and the most common inherited mutation is factor five Leiden (FVL). The latter is due to a single point mutation in factor V, which replaces Arg 506 with Glu residue (Nichols and Heit 1996). FVL represents more than 90% of APCR inherited causes (Nichols and Heit 1996), while the acquired causes of APCR include cancer (Sarig et al. 2005), pregnancy, hormone replacement therapy, and oral contraceptives (Curvers et al. 2002).
The available information about the association between APCR and IVF failure, defined as the inability to reach clinical pregnancy, is quite rare. However, since FVL is responsible for the majority of APCR cases, it is not uncommon to use FVL studies for proper findings validation. Understandably, the results on the impact of FVL on IVF effectiveness are also contradictory and therefore inconclusive (Di Nisio et al. 2011;Tan et al. 2016). Nevertheless, Syrian women with recurrent pregnancy loss had a higher prevalence of APCR (30%) compared to controls (11%) (Alhalaki et al. 2016). This study aimed to investigate the association between APCR and IVF outcomes in Syrian women and the impact of anticoagulation therapy on breaking that probable link.

Materials and methods
In this retrospective cohort study, we reviewed the medical records of females who sought fertility care in 2019 at the Orient fertility centre, which is the largest of its kind in Syria. Only patients who performed an APCR assay at our centre were included. This study was approved by the scientific and ethical committee at the faculty of medicine of Damascus University in September 2019 and by the board of directors at Orient fertility centre.
Blood collection methods, laboratory analysis, and test assays were detailed in a previous study (Alhalaki et al. 2016). Samples collected for oestradiol studies were collected on the third day of menstruation. The assay for APCR used in this study was the STA-STACLOT APC-R Test (Diagnostica Stago, Asnieres, France) which has 99.6% sensitivity, 99.7% specificity, and 99.2% positive predictive value, and 99.9% negative predictive value for FVL. This test is based on the specific activation of factor X by Crotalus viridis helleri snake venom, with the presence of activated protein C. The results are given as clotting time in seconds. According to the manual of the machine, an activated partial thromboplastin time (aPTT) lower than 120 s is considered APCR Positive, while patients with aPTT level equal to or higher than 120 s are considered APCR Negative (Quenhenberger et al. 2000;Alhalaki et al. 2016). APCR Positive patients did not undergo genetic testing for FVL, due to the high costs of these investigations. All known and manageable causes of infertilitysuch as endometrial congestion and polyps-were managed before the time of the IVF.
Patients diagnosed with APCR were treated with aspirin and low-molecular-weight heparin (LMWH) following the hospital protocol. A daily dose of 80 mg of aspirin was prescribed from the day of embryo transfer till the end of the eighth month, and 0.45 mg of LMWH was administered daily from the day of embryo transfer till the end of the pregnancy. However, APCR assay is not routinely done as the Canadian Fertility and Andrology Society guideline did not recommend the testing for thrombophilia in patients with recurrent pregnancy loss (Shaulov et al. 2020). Thus, the assay was only performed when there was a high thrombophilia suspicion or when the IVF attempt failed in infertile women. That is why in some patients, APCR was not managed in advance as shown in Figure 1. The hospital management protocol was in accordance with the Canadian guideline, which recommended restricting the use of anticoagulants in research setting in patients with recurrent implantation failure (Shaulov et al. 2020). So, the treatment was only given after patients' informed consent was obtained. Patients who were under treatment were excluded from the analysis to study the pure effect of APCR on IVF outcomes. All patients underwent controlled ovarian stimulation with a long or antagonist protocol. Pregnancy was confirmed in the lab by measuring Beta-human chorionic gonadotropin and clinically by seeing the gestational sac.
The data were collected from patients' records in the hospital using an electronic form designed on Microsoft Access 365 version 2103 (the year 2021). The eligible records were screened for data on medical, familial, and child-bearing Figure 1. Flow diagram of IVF attempts and APCR assays conducted and their timing. The � sign refers to patients who were lost to follow-up after the embryos transferred and those with unsuccessful ovarian stimulation. The �� sign refers to a patient who was diagnosed after the fourth IVF attempt. history, as well as radiological and laboratory investigations that were indicated for infertility. All IVF attempts were considered for data collection, and information about the protocol used and IVF outcomes was also gathered. Data were imported from Access into the Statistical Package for the Social Sciences version 26.0 (SPSS Inc., Chicago, IL, USA). Categorical variables were represented as frequencies and percentages, and Chi-square or Fisher's exact tests were used to test association among them. As for continuous variables, medians and interquartile ranges were used as the data were not normally distributed. Their associations were investigated using Mann-Whitney U-test. An alpha value of 0.05 was used to determine the threshold of statistical significance.

Results
A total of 552 records were collected; 66 of them had positive APCR (12%). Four hundred thirty-one of them had primary infertility, and 98 had secondary infertility. The median duration of infertility was 6 years, with an interquartile range between three and ten years. The distribution of age, infertility classification, and infertility duration were homogenous among females with and without APCR as shown in Table 1.
In 300 couples (54.3%), male partners contributed to infertility, which was the most common factor for infertility in the sample as shown in the Supporting Information Table S1. Other common factors were, polyps (80; 14.5%), polycystic ovaries (76; 13.8%), and diminished ovarian reserve (72; 13%). Thirty-one percent of the total cases were classified as unexplained infertility. No significant differences were found between the two groups regarding all these factors except for polyps; they were less common in women positive for APCR (1; 1.5%) in comparison to women negative for it (79; 16.3%; p ¼ .001; Supporting Information Table S1).
The second Supporting Information Table shows that 552, 181, 37, and 13 women underwent one, two, three, and four attempts of IVF, respectively. The long protocol for ovarian stimulation was two to twelve times more commonly performed in all IVF attempts. The number of embryos transferred differed, but its median was mostly four for all attempts and subgroups. Data analysis revealed no statistically significant differences between the positive and negative APCR groups in the protocol used or in the number of embryos transferred. Prolactin, TSH, AMH, E2, and LH in the first two IVF attempts were homogenous between the two groups except for prolactin in the first IVF attempt. It was lower in the positive APCR group (median: 15.8; IQR: 10.2-22.0) than in the negative APCR group (median: 19; IQR: 12.0-27.2; p ¼ .04). Cardiolipin IgG (CLG) and Cardiolipin IgM (CLM) were also significantly higher in women with positive APCR with p-values of .024 and .015, respectively (Supporting Information Table S2). Nevertheless, CLG and CLM levels were similar between women who achieved and did not achieve implantation without any significant association (median: 3.1; IQR: 2.6-3.6; p ¼ .792), (median: 1.8; IQR; 1.4-2.8; p ¼ .377, respectively) at the first IVF attempt.
Regarding IVF outcomes, the positive APCR group had only five clinical pregnancies (17.2%) in comparison to 227 clinical pregnancies (54%) in the negative APCR group during the first IVF attempt (p < .001) with an odds ratio of 0.18 (95% CI: 0.07-0.47). However, this statistical difference faded away for live births in the first IVF attempt. Only 21 foetuses reached this stage in the negative APCR group compared to no live births in women positive for APCR as shown in Table 2. Of note, we excluded 20, 13, and 1 positive APCR cases from the first, second, and third IVF attempts presented in Table 2, respectively, because they were on anticoagulant treatment.
The management protocol made a higher clinical pregnancy frequency possible for both the first and second IVF attempts as shown in Table 3. In the first IVF attempt, 18 women (90%) reached clinical pregnancy after treatment   compared to five women (17.2%) without treatment (p < .001) with an odds ratio of 43.2 (95% CI: 7.51-248.6). While in the second IVF attempt, ten treated women (76.9%) reached the same goal compared to none of the untreated women (p ¼ .036). Nevertheless, no live births could be achieved in the APCR-positive group even with anticoagulation.

Discussion
This study illustrated the association between APCR and IVF outcomes in Syrian infertile women. Untreated women with APCR reached fewer clinical pregnancies (p < .001) in the first IVF attempt compared to women without APCR. For the second, third, and fourth IVF attempts, the results were similar to the first IVF, but they did not reach statistical significance. Live births were also lower, but the difference was under powered to reach statistical significance. Women with APCR who underwent treatment with aspirin and LMWH had more clinical pregnancies than women with APCR who did not receive any treatment.
We found an association between APCR and implantation failure, with no significant difference in live births. However, the published reports are inconsistent about the effect of APCR/FVL on pregnancy outcomes. Studies reported an association between APCR and first-trimester abortions (Balasch et al. 1997), early and late miscarriages, and lower live births (Rai et al. 2001). Furthermore, a third study related preclinical pregnancy losses and second-trimester losses to FVL (Tal, et al. 1999), with a higher prevalence of APCR among women with a history of second and third-trimester foetal losses compared to healthy controls (Mello et al. 1999). In addition to, a meta-analysis, that associated FVL with increased early, late recurrent foetal loss, and late non-recurrent foetal loss, it also found that APCR was associated with early recurrent foetal loss (Rey et al. 2003). Another meta-analysis by Di Nisio et al. (2011) reported that ART failure patients had significantly more FVL mutation. On the other hand, a contradicting meta-analysis negated the association between APCR/ FVL, and implantation failure (Tan et al. 2016). This contrast may be due to the low number of studies included, and the lack of embryo aneuploidy screening (Tan et al. 2016). Overall, the discrepancy in study designs, inclusion and exclusion criteria, the lack of a united method to test for thrombophilia, and the lack of a comprehensive screen for all infertility causes might result in incomparable populations and thus contradicting results.
To date, guidelines do not recommend the use of anticoagulants in women with APCR due to the insufficient and contradicting evidence available (Skelley et al. 2017;American College of Obstetricians and Gynecologists 2018;Shaulov et al. 2020). Our results came in favour of the management with promising outcomes, which was in line with the randomised controlled trial by Qublan et al. (2008), who found that using LMWH for thromboprophylaxis in patients with thrombophilia significantly increased implantation pregnancy rate, live birth rate, and decreased the abortion rate. However, their results could not be generalised to APCR patients since only three FVL patients were included (Qublan et al. 2008).
Another study by Aracic et al. (2016) also found that LMWH increased live birth and decreased first-and second-trimesters abortions in thrombophilia patients. On the other hand, aspirin or the combination between LMWH and aspirin yielded similar rates of live birth and miscarriage compared to LMWH alone (Karada� g et al. 2020). This is not the first study to show improvement in IVF outcomes in thrombophilia patients using this regimen, since another meta-analysis by Hamuly� ak et al. (2020) reported that the combination of heparin with aspirin might increase live birth in women with recurrent pregnancy loss and persistent antiphospholipid antibodies.
The prevalence of APCR was 12% in our study, which is in the range of the global prevalence (10%-15%; Winkler 2019). Although risk factors, causes of infertility, and lab tests were homogeneous among the positive and negative APCR women, some differences reached statistical significance. First, polyps were found less in APCR-positive patients, suggesting that APCR can possibly reduce endometrial polyps' formation. Second, antiphospholipid antibodies were higher in APCR patients, which agreed with Arachchillage et al. (2014), who reported that antiphospholipid syndrome causes acquired APCR. However, the debate remains about the exact association between antiphospholipid antibodies and APCR (Gardiner et al, 2006;Arachchillage et al. 2014).
To our knowledge, this study is the first to investigate the effect of aspirin and LMWH on implantation specifically in APCR patients. Although the low live birth count limited our conclusions, it can be attributed to the loss of follow-up in many patients after achieving clinical pregnancy, because many of them lived in remote areas, with harder mobility due to the Syrian war. In addition, women with low thrombophilia suspicion or who had a successful IVF attempt were not screened for APCR. Thus, the results might be prone to selection bias, since those women might include positive APCR cases that escaped the screening. Rare thrombophilia such as protein S, protein C deficiency, and prothrombin gene was not investigated due to the high cost of the corresponding tests. Nevertheless, our sample of APCR cases was still larger than other studies giving it appropriate power for the test reported. Controlled clinical trials with more restricted inclusion-exclusion criteria and more robust followup are needed to confirm our findings.
In conclusion, APCR is associated with fewer clinical pregnancies in infertile women undergoing IVF. However, aspirin and LMWH can mitigate this effect. These two findings might support the recommendation to routinely perform APCR in infertile women before IVF attempts and to prescribe anticoagulation (aspirin and LMWH) for positive cases.