Eirini Papadimitriou1,2, Georgios Boutzios1, Alexander G Mathioudakis3,4, Nikos F Vlahos5, Panayiotis Vlachoyiannopoulos1, George Mastorakos2. 1. Department of Pathophysiology, Laiko University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece. 2. Endocrine Unit, Aretaieion University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece. 3. Division of Infection, Immunity and Respiratory Medicine, School of Biomedical Sciences, The University of Manchester, Manchester, United Kingdom. 4. North West Lung Centre, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom. 5. 2nd Department of Obstetrics and Gynecology, Aretaieion University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
Abstract
PURPOSE: A systematic review and meta-analysis was conducted comparing the presence of anti-phospholipid (anti-PL) antibodies between women of reproductive age, without diagnosis of antiphospholipid syndrome, who experienced at least two implantation failures following in vitro fertilization and embryo transfer (IVF-ET), and either women who had a successful implantation after IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. METHODS: Systematic search of the literature and meta-analysis of the relevant studies studying presence of antiphospholipid antibodies in women experiencing at least two implantation failures in IVF-ET as compared to either women who had a successful implantation after IVF-ET or/and women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Six hundred ninety-four published reports were retrieved; 17 of them fulfilled the inclusion criteria set. RESULTS: Presence of either any type of anti-phospholipid or anticardiolipin antibodies or lupus-anticoagulant in women experiencing at least two implantation failures in IVF-ET was associated with increased implantation failure compared to women who had a successful implantation after IVF-ET (relative risk, RR: 3.06, 5.06 and 5.81, respectively). Presence of either anticardiolipin or lupus-anticoagulant or anti-beta2 glycoprotein-I or anti-phosphatidylserine antibodies in women experiencing at least two implantation failures in IVF-EΤ was associated with increased implantation failure compared to unselected healthy fertile women with no history of IVF-ET (RR:13.92, 6.37, 15.04 and 164.58, respectively). CONCLUSION: The prevalence of antiphospholipid antibodies, particularly that of anti-beta2 glycoprotein-I and anti-phosphatidylserine antibodies, in women experiencing at least two implantation failures in IVF-ET without diagnosis of antiphospholipid syndrome is significantly greater than either in women who had a successful implantation after IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. TRIAL REGISTRATION NUMBER: PROSPERO ID: CRD42018081458.
PURPOSE: A systematic review and meta-analysis was conducted comparing the presence of anti-phospholipid (anti-PL) antibodies between women of reproductive age, without diagnosis of antiphospholipid syndrome, who experienced at least two implantation failures following in vitro fertilization and embryo transfer (IVF-ET), and either women who had a successful implantation after IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. METHODS: Systematic search of the literature and meta-analysis of the relevant studies studying presence of antiphospholipid antibodies in women experiencing at least two implantation failures in IVF-ET as compared to either women who had a successful implantation after IVF-ET or/and women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Six hundred ninety-four published reports were retrieved; 17 of them fulfilled the inclusion criteria set. RESULTS: Presence of either any type of anti-phospholipid or anticardiolipin antibodies or lupus-anticoagulant in women experiencing at least two implantation failures in IVF-ET was associated with increased implantation failure compared to women who had a successful implantation after IVF-ET (relative risk, RR: 3.06, 5.06 and 5.81, respectively). Presence of either anticardiolipin or lupus-anticoagulant or anti-beta2 glycoprotein-I or anti-phosphatidylserine antibodies in women experiencing at least two implantation failures in IVF-EΤ was associated with increased implantation failure compared to unselected healthy fertile women with no history of IVF-ET (RR:13.92, 6.37, 15.04 and 164.58, respectively). CONCLUSION: The prevalence of antiphospholipid antibodies, particularly that of anti-beta2 glycoprotein-I and anti-phosphatidylserine antibodies, in women experiencing at least two implantation failures in IVF-ET without diagnosis of antiphospholipid syndrome is significantly greater than either in women who had a successful implantation after IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. TRIAL REGISTRATION NUMBER: PROSPERO ID: CRD42018081458.
Infertility is a public health problem which affects 1:10 women of reproductive age [1, 2]. Its estimated world prevalence is 186 million people [1, 2]. Assisted reproductive technology (ART) has led to a significant rise in live births following the introduction of in vitro fertilization (IVF) [3]. Over 1,250,000 ART cycles, resulting in birth of over 225,000 babies, were reported by 2,419 clinics globally in 2007. The availability of ART varies by country, from 12 to 4,140 treatments per million population [4].Rheumatic diseases can affect quality of life and reproduction. Pregnancy complications are increased in patients with systemic lupus erythematosus and antiphospholipid syndrome (APS). The latter is an autoimmune acquired thrombophilia, which occurs either alone or in combination with other autoimmune diseases, mainly with systemic lupus erythematosus [4]. Antiphospholipid antibodies represent a heterogeneous group of antibodies, which recognize various phospholipids, phospholipid-binding proteins, and phospholipid protein complexes. Clinical manifestations of APS include fertility problems and pregnancy complications (such as repeated miscarriages) as well as venous or arterial thrombosis [5]. Evaluation of circulating anti-phospholipid (anti-PL) antibodies is part of the serological work-up following miscarriage. When circulating anti-PL antibodies are positive at initial diagnosis, testing should be repeated at least 12 weeks later to confirm diagnosis of APS [5]. According to revised Sapporo criteria, diagnosis of APS takes into account lupus anticoagulant (LA), anti-cardiolipin (anti-CL) antibodies or anti-β2glycoprotein I (anti-β2GP I) antibodies of either IgG or IgM isotype.The relationship between presence of anti-PL antibodies (without diagnosis of APS) and implantation failure has been examined by several original studies which suggested that presence of anti-PL antibodies, even without diagnosis of APS, impairs implantation. Antiphospholipid antibodies, especially anti-beta2 glycoprotein I (anti-β2GPI) antibodies, in pregnancy, appear to act directly on trophoblasts by activating pro-apoptotic and pro-inflammatory mechanisms [6]. At the same time, thrombosis of placental chorionic arteries and activation of the complement system intravascularly lead to the cell death of the trophoblast by decreasing trophoblast viability, syncytialization, and capacity for invasion [6].Whether the presence alone of anti-PL antibodies in healthy women of reproductive age who do not fulfill the criteria for APS, might affect implantation and embryo transfer (ET) following IVF, is not decided as yet in the literature. To fill this gap this systematic review and meta-analysis were conducted.
Material and methods
Protocol
Search strategy and selection of studies
This systematic review and meta-analysis was based on a protocol registered prospectively in PROSPERO database for systematic review protocols (ID: CRD42018081458) and follows Preferred reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [7, 8]. The electronic databases of Medline (Pubmed) and Cochrane library were reviewed systematically from inception to April 2021, using appropriate controlled vocabulary and free search terms to identify studies evaluating fertility in women in association with presence of any type of anti-PL antibodies (detailed search strategy is available in the online Appendix 1 in S1 Appendix). Titles, abstracts and full text (when appropriate) of all identified studies were screened for eligibility by one author (E.P.). The same author extracted from the studies the following pieces of information in a pre-specified standardized MS Excel: full reference; study identifiers; study design; eligibility; predefined outcomes; number of participants (population index and controls); characteristics of participants; details on the outcomes of interest. The term population index refers to the total number of women who experienced at least two implantation failures after IVF-ET. Search strategy was validated by GM. When EP raised a discrepancy, GM was consulted. The extracted characteristics of participants were: age; cause of subfertility wherever applicable; number of years of subfertility wherever applicable; numbers of IVF/ET attempts wherever applicable; number of retrieved and fertilized oocytes; quality of embryos (defined as regular blastomeres, or according to the presence of even cleavage; even cell sizes; less than 20% fragmented blastomeres); number of transferred embryos wherever applicable; past medical history of women included in each study was retrieved (no women suffered from APS or had a history of thrombosis); time period in which participants were enrolled; provenance of participants; laboratory technique for measurement of any type of anti-PL antibodies. All these steps were validated by a second reviewer (A.G.M.). Disagreement was resolved by discussion or adjudication by a third investigator if necessary (G.M.)
Criteria for inclusion of studies in the meta-analysis
Studies fulfilling all of the following criteria were included in this meta-analysis:Studies published in English with prospective or retrospective observational design.All study populations should be consisted by healthy women of reproductive age not suffering from any known autoimmune, endocrine or infectious diseases.Studies comparing the prevalence of any type of anti-PL antibodies between women experiencing at least two implantation failures in IVF-ET (population index) vs. either women experiencing one successful IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Studies with control women experiencing one successful IVF-ET were included in subgroup A of selected studies [9-14], while studies with control women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET were included in subgroup B of selected studies [3, 9, 13, 15–24].
Study outcomes extracted for the meta-analysis
The primary outcome extracted from the selected studies was presence or not of any type of anti-PL antibodies. Secondary outcomes extracted from the selected studies were presence or not of: anticardiolipin (anti-CL), lupus anticoagulant (LA) and anti-β2GPI antibodies (all three representing aPL included in the Sapporo criteria for APS diagnosis), as well as anti-phosphatidylserine (anti-PS), anti-phosphatidylcholine (anti-PC), anti-phosphatidylethanolamin (anti-PE), anti-phosphatidylinositol (anti-PI), anti-phosphatidylglycerol (anti-PG) and anti-phosphatidic acid (anti-PA) antibodies which have gained importance in recent literature for APS diagnosis.
Risk of bias assessment
Risk of bias was assessed by two authors independently (E.P. and A.G.M.) by employing the Newcastle-Ottawa Scale [25]. Disagreement was resolved by discussion or adjudication by a third investigator if necessary (G.M.). In line with the previously submitted protocol of the meta-analysis, a publication bias analysis (Funnel plot and Egger’s) was not performed because comparisons consisted of less than 25 eligible studies render such analysis less informative [26].
Statistical analyses
Heterogeneity among selected studies was evaluated in each analysis, using I2 statistic [26, 27]. According to Cochrane handbook: I2 ≥ 75%, I2 between 75% and 50% or I2 ≤ 50% reflect substantial, significant or non-significant heterogeneity among the selected studies, respectively [27]. When I2 was ≥ 75%, possible causes of heterogeneity were investigated by performing pre-specified subgroup analyses. Meta-analysis was not performed in case of substantial heterogeneity which could not be resolved by subgroup analysis. In these cases, findings were reported narratively.All outcomes were dichotomous and were analyzed by calculating relative risks (RR) and 95% confidence intervals (CI). For data synthesis the random effect model was employed as significant clinical and methodological heterogeneity among the included studies was anticipated. In addition, a priori specified subgroup analyses were conducted to explore significant or substantial heterogeneity and to further evaluate the soundness of results.In a pre-specified sensitivity analysis, meta-analyses were repeated using the fixed effects model. All analyses were performed using Review Manager 5.3 Software [28].
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Results
Search strategy for the systematic review
Search strategy identified 621 and 73 references in Medline and Cochrane Library, respectively (Fig 1). After removal of duplicate records between the two databases, a total of 629 studies were screened by title and abstract. Of these references, 40 were deemed potentially eligible because they reported studies regarding healthy women of reproductive age with implantation failure after IVF-ET and were assessed by full-text. Twenty-three of these 40 studies were excluded because either the outcome was not precisely reported or their design did not fulfill the inclusion criteria set. In the remaining 17 studies included in the present systematic review and meta-analysis (ten and seven studies were retrospective and prospective cohort studies, respectively), 4,075 healthy women of reproductive age were evaluated. Selection process is described in Fig 1, while the characteristics of each of the included studies are reported in the online Appendix 2 in S1 Appendix.
Fig 1
PRISMA flow diagram of the selection process from identified studies to selected studies through Medline and Cochrane Library.
Characteristics of the women in the selected studies
The women with implantation failure [absence of positive pregnancy tests based on beta human chorionic gonadotropin (hCG) evaluation; in none of the studies the timing of hCG measurement was reported] after IVF-ET included in all studies had at least two implantation failures (range of failures: 2–6) following transfer of good quality embryos. Three studies included confirmation of a gestational sac by ultrasound three weeks after embryo transfer [9, 16, 18]. All women included in these studies were not receiving any additional treatment.
Subgroup A of selected studies
Studies of this subgroup, included women referred for IVF-ET due to similar indications [9-14]. Unexplained infertility was the predominant indication, followed by tubal factor -for the majority of them. Age, duration and type of infertility did not differ between population index and controls studied in this subgroup.
Subgroup B of selected studies
In these studies, the majority of patients received IVF for unexplained infertility. In studies of this subgroup, other, not frequently encountered, indications for IVF-ET were endometriosis, ovulation disorders or mixed infertility.
Characteristics of the included studies in the systematic review
Ovarian stimulation protocols employed
Four studies reported that a standard protocol for ovarian stimulation was followed (a combined regimen of GnRH agonists and human menopausal gonadotrophins) [9–11, 18]. The remaining studies did not report the specific ovarian stimulation protocol employed.
Assays employed for the antibodies evaluation
All selected studies employed enzyme-linked immunosorbent assays (ELISA) for presence of any type of anti-PL antibodies. Lupus anticoagulant was evaluated either by the kaolin cephalin clotting time utilizing sensitive reagents or by the dilute Russell’s viper venom time with a neutralization procedure using frozen–thawed platelets or by both techniques. Results for anti-PL and LA antibodies were expressed as positive or negative. Diagnostic cut-offs for the antibodies were reported in: Qublan H. et al. (anti-CL antibodies positive >10 IU/ml; qualitative positivity or negativity for LA) [13]; Vaquero E. et al. (anti-CL antibodies positive >20; qualitative positivity or negativity for LA) [18]; Bellver et al. (anti-CL antibodies positive >20 gPL/ml or mPL/ml for IgG or IgM isotype, respectively; qualitative positivity or negativity for LA) [21]; Sanmarco M. et al. [antibodies positive for anti-CL: IgG ≥20 GPLU; for anti-β2GPI IgG ≥10 B2GU; for aPE IgG ≥15 PEGU (GPLU, MPLU, B2GU and PEGU are arbitrary units for optical density)] [20]; Geva E. et al. (anti-CL antibodies positive >23 GPLU) [12].In six studies positivity was based on optical density measurement exceeding the 99th or the 95th percentile of measurements established for each phospholipid in healthy individuals of reproductive age [11, 14, 17, 19, 22, 23]. Six studies did not report diagnostic cut-offs [3, 9, 10, 15, 16, 24]. Manufacturers of the assays are reported in all studies except two [13, 20].Six studies (n = number of women studied; n = 438) evaluated presence of any type of anti-PL antibodies (anti-CL, anti-PS, anti-PC, anti-PE, anti-PI and anti-β2GPI antibodies) [3, 9–13]. Four studies (n = 360) evaluated presence of anti-CL antibodies [9, 11–13]. Two studies (n = 268) evaluated presence of LA [9, 13]. One study (n = 42) evaluated presence of anti-PS, anti-PΙ, anti-PC, anti-PE, anti-PA and anti-PG antibodies [13]. Meta-analysis was performed for the prevalence of either any type of anti-PL antibodies or for anti-CL antibodies or for LA antibodies.Thirteen studies (n = 3,637) evaluated the presence of any type of anti-PL antibodies [3, 9, 13, 16–24].Six studies (n = 2610) evaluated the presence of anti-CL antibodies [9, 12, 14, 18, 20, 21]. Three studies (n = 2004) evaluated the presence of anti-CL-IgG as well as anti-CL-IgM antibodies [18, 20, 21]. Four studies (n = 353) evaluated the presence of LA antibodies [9, 12, 15, 20]. Three studies (n = 2144) evaluated the presence of anti-β2GPΙ [9, 18, 22]. Two studies (n = 1978) evaluated the presence of anti-PS antibodies [18, 21]. One study (n = 1926) evaluated the presence of anti-PI, anti-PA, anti-PE and anti-PG antibodies [18]. Meta-analysis was performed for the prevalence of either anti-CL-IgG or LA or anti-β2GPI or anti-PS antibodies.Qublan et al. and Khizroeva et al. evaluated women with implantation failures after IVF-ET compared with both control groups. Therefore, these studies are included in both subgroups A and B of selected studies [9, 13].Data regarding antibodies studied only in one study in each subgroup were not included in a meta-analysis. Thus, neither data on anti-PΙ, anti-PC, anti-PE, anti-PA, anti-PG and anti-PS antibodies reported only in the study by Kaider et al. (Subgroup A) nor data on anti-PΙ, anti-PE, anti-PA and anti-PG antibodies reported only in the study by Ulcova-Gallova et al. (Subgroup B) were included in a meta-analysis (Tables 1 and 2).
Table 1
Prevalence (reported as percentages in parentheses) of different types of anti-phospholipid (anti-PL) antibodies in studies included in subgroup A of studies.
any type of anti-PL
Anti-CL
LA
anti-PΙ
anti-PC
anti-PE
anti-PA
anti-PG
anti-PS
Birkenfeld et al.
18/56 (32.1%) vs 0/14 (0%)
Buckingham et al.
5/22 (22.7%) vs 13/71(18.3%)
Geva et al.
3/50 (6%) vs 0/40 (0%)
3/50 (6%) vs 0/40 (0%)
Qublan et al.
17/90 (18.9%) vs 4/90 (4.4%)
9/90 (10%) vs 2/90 (2.2%)
8/90 (8.9%) vs 2/90 (2.2%)
Khizroeva et al.
75/178(42.1%) vs 22/169 (13%)
16/178 (9%) vs 3/169 (1.8%)
35/178 (19.7%) vs 5/169 (3%)
Kaider et al.
11/42 (26.2%) vs 0/42 (0%)
3/42 (7.1%) vs 0/42(0%)
3/42 (7.1%) vs 0/42(0%)
12/42 (21.4%) vs 0/42(0%)
3/42 (7.1%) vs 2/42(4.8%)
3/42(7.1%) vs 0/42(0%)
2/42(4.8%) vs 0/42(0%)
1/42 (2.4%) vs 0/42(0%)
Footnote: Studies in subgroup A compare women with at least two implantation failures in IVF-ET vs. women with one successful IVF-ET. Anti-PL: anti-phospholipid antibodies; anti-CL: anti-cardiolipin, antibodies; LA: lupus anticoagulant; anti-PI: anti-phosphatidylinositol antibodies; anti-PC: anti-phosphatidylcholine antibodies; anti-PE: anti-phosphatidylethanolamin antibodies; anti-PA: anti-phosphatidic acid antibodies; anti-PG: anti-phosphatidylglycerol antibodies and anti-PS: antiphospatidilserine antibodies.
Table 2
Prevalence (reported as percentages in parentheses) of different types of anti-phospholipid (anti-PL) antibodies in studies included in subgroup B of studies.
any type of anti-PL
anti-CL
anti-CL Ig-G
anti-CL Ig-M
LA
anti- β2GP I
anti-PI
anti-PE
anti-PA
anti-PG
anti-PS
Alves et al.
48/52 (92%) vs 0/28 (0%)
50/52 (96.2%) vs 0/28 (0%)
29/52 (55.8) vs 0/28 (0%)
50/52 (96%) vs 0/28 (0%)
48/52 (92.3) vs 0/42 (0%)
Bellver et al.
8/57 (14%) vs 6/32 (18.8%)
8/57 (14%) vs 6/32 (18.8%)
1/26 (3.8%) vs 0/32 (0%)
1/26 (3.8%) vs 6/32 (18.8%)
3/26 (11.5%) vs 0/32 (0%)
Coulam 97 et al.
69/312 (22%) vs 5/100 (5%)
13/312 (4.2%) vs 0/100 (0%)
Coulam 02 et al.
34/122 (27.9%) vs 7/107 (6.5%)
Paulmyer-Lacroix et al.
8/40 (20%) vs 1/100 (1%)
5/40 (12.5%) vs 1/100 (1%)
Qublan et al.
17/90 (18.9%) vs 9/100 (9%)
9/90 (10%) vs 3/100 (3%)
8/90 (8.9%) vs 2/100 (2%)
Sanmarco et al.
40/101 (39.6%) vs 8/160 (5%)
Steinvil et al.
17/509 (3.3%) vs 30/637 (4.7%)
Stern et al.
30/105 (28.6%) vs 16/106 (15.1%)
Ulcova-Gallova et al.
928/1926 (48.2%) vs 5/391 (1.3%)
421/1926 (21.9%) vs 8/391 (2%)
349/1926 (18.6%) vs 5/391 (1.3%)
72/1926 (3.7%) vs 3/391 (0.8%)
209/1926 (10.9%) vs 0/391 (0%)
613/1926 (31.8%) vs 0/391 (0%)
377/1926 (19.6%) vs 2/391(0.5%)
240/1926 (12.5%) vs 0/391 (0%)
318/1926 (16.5%) vs 4/391(1%)
778/1926 (40.4%) vs 0/391 (0%)
Vaquero et al.
8/59 (13.6%) vs 0/20 (0%)
3/59 (5%) vs 0/20 (0%)
Khizroeva et al.
75/178 (42.1%) vs 3/80 (3.8%)
16/178 (9%) vs 1/80 (1.3%)
35/178 (19.7%) vs 1/80 (1.3%)
56/178 (31.5%) vs 3/80 (3.8%)
Saxtorph et al. 2020
2/86 (2%) vs 0/37 (0%)
Footnote: Subgroup B compares women with at least two implantation failures in IVF-ET vs. either women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Anti-PL: anti-phospholipid antibodies; anti-CL: anti-cardiolipin, antibodies; LA: lupus anticoagulant; anti- β2GPI: anti-β2glycoprotein I antibodies; anti-PI: anti-phosphatidylinositol antibodies; anti-PE: anti-phosphatidylethanolamin antibodies; anti-PA: anti-phosphatidic acid antibodies; anti-PG: anti-phosphatidylglycerol antibodies and anti-PS: anti-phospatidilserine antibodies.
Footnote: Studies in subgroup A compare women with at least two implantation failures in IVF-ET vs. women with one successful IVF-ET. Anti-PL: anti-phospholipid antibodies; anti-CL: anti-cardiolipin, antibodies; LA: lupus anticoagulant; anti-PI: anti-phosphatidylinositol antibodies; anti-PC: anti-phosphatidylcholine antibodies; anti-PE: anti-phosphatidylethanolamin antibodies; anti-PA: anti-phosphatidic acid antibodies; anti-PG: anti-phosphatidylglycerol antibodies and anti-PS: antiphospatidilserine antibodies.Footnote: Subgroup B compares women with at least two implantation failures in IVF-ET vs. either women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Anti-PL: anti-phospholipid antibodies; anti-CL: anti-cardiolipin, antibodies; LA: lupus anticoagulant; anti- β2GPI: anti-β2glycoprotein I antibodies; anti-PI: anti-phosphatidylinositol antibodies; anti-PE: anti-phosphatidylethanolamin antibodies; anti-PA: anti-phosphatidic acid antibodies; anti-PG: anti-phosphatidylglycerol antibodies and anti-PS: anti-phospatidilserine antibodies.Based on the Newcastle Ottawa scale all studies of subgroup A were rated as of low risk in all assessed domains: selection bias of population index and control, performance bias, detection bias and attrition bias.Ten out of thirteen studies of subgroup B (77%) were rated as having low risk and three (23%) as having unclear risk of bias regarding selection of population index. Regarding selection of controls, five out of thirteen studies of subgroup B (39%) were rated with unclear risk of bias. All studies of subgroup B were rated with low risk of bias regarding performance bias, detection bias and attrition bias. Detailed assessment of the risk of bias is available in online Appendix 3 in S1 Appendix.
Outcomes of systematic review and meta-analysis
The reported results are the outcome of separate random effect analyses of subgroup A and subgroup B. The sensitivity analyses using fixed effect methods showed similar results.The majority of selected studies supported the association between presence of any type of anti-PL antibodies and infertility. Studies included in subgroup A were fairly homogenous (I² range: 0–15%) and provided quite reliable results, whereas studies included in subgroup B were less homogeneous.
Outcomes of subgroup A of selected studies (Table 1)
Twenty-nine percent and 9.6% of population index and controls respectively, reported presence of any type of anti-PL antibodies among anti-CL, anti-β2GPI, anti-PS, anti-PC, anti-PE, anti-PI, anti-PA and anti-PG antibodies of IgG, IgM or IgA isotypes (six studies) [3, 9–13]. Population index showed a RR for the presence of any type of the above mentioned anti-PL antibodies of 3.06 for implantation failure (95% CI: 1.97, 4.77, I² = 15%) compared to controls (Fig 2). Because the anti-PC and anti-PE antibodies have lost part of their importance in the recent literature, a meta-analysis has been also performed without including the only study which evaluated these antiphospholipid antibodies. The obtained RR did not change substantially [RR: 2.89 (95% CI: 1.73, 4.81), I2 = 22%]. In addition, 8.6% and 1.5% of population index and controls, respectively, had present anti-CL antibodies (four studies) [9, 11–13]. Population index showed a RR for the presence of anti-CL antibodies of 5.06 for implantation failure (95% CI: 2.14, 11.95; I² = 0%) compared to controls (Fig 3). Moreover, 16% and 2.7% of population index and controls, respectively, had LA antibodies present (two studies) [9, 12]. Population index showed a RR for presence of LA antibodies of 5.81 for implantation failure (95% CI: 2.66, 12.71; I² = 0%) compared to controls (Fig 4).
Fig 2
Meta-analyses assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup A between women with at least two implantation failures in IVF-ET (population index) vs. women with one successful IVF-ET (control).
Fig 2: meta-analysis assessing the risk for implantation failure in relation to the presence or not of any type of anti-PL antibodies.
Fig 3
Meta-analyses assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup A between women with at least two implantation failures in IVF-ET (population index) vs. women with one successful IVF-ET (control).
Fig 3: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL antibodies.
Fig 4
Meta-analyses assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup A between women with at least two implantation failures in IVF-ET (population index) vs. women with one successful IVF-ET (control).
Fig 4: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies. +Abs: positive antibodies, total #: total number of participants.
Meta-analyses assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup A between women with at least two implantation failures in IVF-ET (population index) vs. women with one successful IVF-ET (control).
Fig 2: meta-analysis assessing the risk for implantation failure in relation to the presence or not of any type of anti-PL antibodies.Fig 3: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL antibodies.Fig 4: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies. +Abs: positive antibodies, total #: total number of participants.
Outcomes of subgroup B of selected studies (Table 2)
Three studies out of six in subgroup B point out two distinct aCL isotypes (aCL/Ig-G, aCL/Ig-M) in their reporting of aCL evaluation. However, meta-analysis was conducted only for the aCL/Ig-G isotype because, for this isotype, a non-significant heterogeneity among the selected studies was observed, whereas for the aCL/Ig-M isotype the heterogeneity observed was substantial. Thus, in line with the methodology and guidance of Cochrane handbook, a meta-analysis was not meaningful for the aCL/Ig-M isotype. Nineteen percent and 1% of population index and controls, respectively had anti-CL-IgG antibodies present (three studies) [18, 20, 21]. Presence of anti-CL antibodies of the IgG isotype was more strongly associated with implantation failure after IVF-ET compared to that of the IgM isotype. Specifically, population index showed a RR for the presence anti-CL antibodies of the IgG isotype of 13.92 for implantation failure (95%CI: 6.21, 31.21; I2 = 0%) compared to controls (Fig 5).
Fig 5
Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control).
Fig 5: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL-IgG antibodies.
Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control).
Fig 5: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL-IgG antibodies.Fourteen percent and 1.3% of population index and controls, respectively, had LA antibodies present (four studies) [9, 12, 15, 20]. Population index showed a RR for the presence of LA antibodies of 6.37 for implantation failure (95% CI: 2.25, 18.04; Ι2 = 0%) compared to controls (Fig 6).
Fig 6
Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control).
Fig 6: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies.
Fig 6: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies.Almost thirteen percent and 0.7% of population index and controls, respectively, had anti-β2GPI antibodies present (three studies) [9, 18, 22]. Population index showed a RR for the presence of anti-β2GPI of 15.04 for implantation failure (95% CI: 3.47, 65.10; Ι2 = 44%) compared to controls (Fig 7).
Fig 7
Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control).
Fig 7: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti- β2GPI antibodies.
Fig 7: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti- β2GPI antibodies.Two studies assessed anti-PS antibodies [18, 21]. Forty-two percent of population index had anti-PS antibodies present, as compared 0% of controls. Population index showed a RR for the presence of anti-PS of 164.58 for implantation failure (95% CI: 23.31, 1162.26; I2 = 0%), compared to controls (Fig 8).
Fig 8
Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control).
Fig 8: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PS antibodies. +Abs: positive antibodies, total #: total number of participants.
Fig 8: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PS antibodies. +Abs: positive antibodies, total #: total number of participants.Two studies of this subgroup did not show any association between the presence of any type of anti-PL or anti-CL antibodies and IVF-ET outcome [3, 21]. However, the results of both studies should be examined with caution as Bellver et al. were based on a very limited study population, while it is unclear how Steinvil et al. selected their control group.
Discussion
Among 629 references that this systematic search yielded from Medline and Cochrane Library, a limited number of 17 studies, involving 4,075 women of reproductive age, were included in this systematic review and meta-analysis. The addition of good studies in the future will improve the accuracy of the deduced conclusions. All included studies involved women with at least two implantation failures in IVF-ET vs. either women with one successful IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. When a specific type of anti-PL antibodies was evaluated in heterogeneous or single studies, the reported data were not included in the meta-analysis.We found, in this meta-analysis, that in women experiencing at least two implantation failures in IVF-ET, presence of either any type of anti-PL antibodies or anti-CL antibodies only or LA antibodies is associated with a significant 3.06, 5.06 and 5.81 RR for impaired implantation rate, respectively, as compared to women experiencing one successful IVF-ET. In addition, in women experiencing at least two implantation failures in IVF-ET, presence of either anti-CL or LA or anti-β2GPI or anti-PS antibodies is associated with a significant 13.92, 3.37, 15.04 and 164.58 RR for impaired implantation rate, respectively, as compared to women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Most importantly, presence of either anti-β2GPI or the rarely evaluated anti-PS antibodies in 13% and 41%, respectively, of all women experiencing at least two implantation failures in IVF-ET and in virtually none of control subjects suggests that these may be very accurate biomarkers (more accurate than the more frequently measured anti-PL antibodies) and urges further evaluation of their potential clinical use in infertility, as well as in APS in general. This is the first meta-analysis evaluating presence of any type of anti-PL antibodies in women experiencing implantation failure in IVF-ET. The results from the 17 selected studies were consistent, of strong methodological quality and support an association between recurrent implantation failure in IVF-ET and presence of anti-PL antibodies. This meta-analysis looked into multiple anti-PL antibodies, including newer markers (i.e. anti-PS antibodies) aiming at reporting a quantitative result based on the homogeneity and similarity in the findings of the included studies. Both in the retrospective and prospective studies included in the present meta-analysis the compared population indices and control groups are well defined. Thus, the accuracy and reliability of the extracted results cannot be limited in either case. The main limitation of this meta-analysis is the lack of relevant prospective, controlled studies and the resulting small number of included studies. Despite the small number of included studies, the conclusions of the present meta-analysis can be considered scientifically valid due to the lege artis mathematical approach followed in this meta-analysis and the homogeneity of the included studies.Except the above mentioned anti-PL antibodies, which were evaluated by meta-analysis, this systematic review pointed towards other types of anti-PL antibodies, which seem to impair implantation and consequently fertility. Presence of specific types of anti-PL antibodies (i.e. anti-PS, anti-PC, anti-PI and anti-PA antibodies) was significantly increased in women with at least two implantation failures in IVF-ET and was associated with increased implantation failure rates, although specific types of anti-PL antibodies are in debate in the literature. Of note, these antibodies were not present in the respective control groups (Tables 1 and 2). The prevalence of these specific types of anti-PL antibodies reaches 40.4% among women with at least two implantation failures in IVF-ET compared to controls (range of prevalence among population index 2.4%-40.4%). Prevalence of anti-PE and anti-PG antibodies was also increased in women with at least two implantation failures in IVF-ET (range among population index 7.1%-19.6%) compared to their respective controls (range among controls: 0–4.8%) (Tables 1 and 2). It is noteworthy that the aforementioned types of anti-PL (anti-PS, anti-PC, anti-PI and anti-PA anti-PE and anti-PG antibodies) are rarely evaluated in clinical practice. One might suspect that they might be positive where other more often evaluated types of anti-PL such as anti-CL, LA or anti-β2GPI antibodies are negative. Of note, in this meta-analysis, were included not only studies reporting anti-PL antibodies included in the revised Sapporo criteria (LA, anti-CL, anti-β2GP I) but also anti-PL antibodies (anti-PS, anti-PC, anti-PE, anti-PI, anti-PG, anti-PA) which have gained importance for APS diagnosis in recent literature. Thus, it should be investigated whether these antibodies, not routinely measured, are involved as well in pathophysiologic aspects of implantation. Thus, questions emerge about their involvement in the underlying pathophysiological mechanisms in implantation.The possible association of anti-PL antibodies with female infertility has been suggested since 1980s. Women with APS and women with anti-PL antibodies may present with impaired ovarian follicles reserve and more frequently with premature ovarian failure [29-31]. Α retrospective study suggested that the presence of APS or just anti-PL antibodies are frequently encountered acquired risk factors for recurrent pregnancy loss and that they are associated with increased risk for ischemic placental dysfunction, such as fetal growth restriction, preeclampsia, premature birth and intrauterine death [32]. Till now, little is known about the biological mechanisms involved in the recurrent ET failures observed in presence of anti-PL antibodies as well as in APS. Anti-PS antibodies bind to human trophoblast in a dose-dependent way affecting thus, trophoblast invasiveness and differentiation of cytotrophoblast into a syncytium. It is also shown that, specifically anti-PS and not anti-CL antibodies are responsible for the decrease of hCG production by the placenta [33]. In vitro and in vivo studies, have suggested that anti-PL antibodies might affect negatively conception, implantation as well as early and recurrent miscarriages. They can impair spontaneous as well as IVF-ET implantation as they are directed against negatively charged phospholipids located in the blood vessels of the uterine mucous membrane, or on the surface of oocytes, or they can affect the early embryo at the initial implantation process [32]. The latter and the maintenance of pregnancy in its early stages could be affected by inhibition of prostaglandin synthesis caused by anti-PL antibodies [32]. The latter affect maternal blood vessels, decidua and trophoblasts. They have been suggested to target tissue plasminogen activator, plasmin, annexin A2 and thrombin [34]. Prothrombin and β2GPI mediate binding of anti-PL antibodies to target cells such as endothelial cells, monocytes, platelets and trophoblasts leading to thrombosis of placental vessels and fetal loss [35, 36]. Anti-PL antibodies and particularly β2GPI-mediated anti-PL antibodies bind to trophoblast monolayers and can induce direct cellular injury, inhibition of proliferation and syncytia formation, apoptosis, and defective invasiveness [35, 36]. In mice, β2GPI is essential for a successful pregnancy and for optimal placental development [12]. In vitro studies indicate that anti-CL antibodies inhibit trophoblast proliferation possibly by a prostacyclin-thromboxane A2 imbalance [37, 38]. Patients’ anti-PS antibodies when co-cultured with rats embryos delay the development of rat yolk sacs [35]. These antibodies have been shown to affect negatively implantation in rats while they bind to human trophoblast in a dose dependent way affecting thus, trophoblast invasiveness and differentiation of cytotrophoblast into a syncytium [33]. To our knowledge, the presence of anti-PC, anti-PE, anti-PI, anti-PG, or anti-PA antibodies has not as yet been associated directly with implantation failure.In summary, this meta-analysis has shown that presence of any type of anti-PL antibodies is associated with impaired implantation among women experiencing at least two implantation failures in IVF-ET and not suffering from APS compared to either women experiencing one successful IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. Presence of anti-β2GPI and anti-PS antibodies suggests an excessive risk. Importantly, types of anti-PL, not frequently measured in daily medical practice (anti-PS, anti-PC, anti-PE, anti-PI, anti-PA and anti-PG antibodies), seem to be stronger predictors of implantation failure in IVF-ET. In women not suffering from APS, presence of antibodies (lupus anticoagulant, anti-cardiolipin antibodies and anti-beta2-glycoprotein I antibodies) included in the definition of APS, seem to be strongly associated with implantation failure in IVF-ET. In guidelines, it is suggested to evaluate anti-PL antibodies in women suffering from recurrent miscarriages [5]. Similarly, in women presenting multiple implantation failures in IVF-ET, it could be suggested to measure these antibodies, in order to investigate causality and, eventually, suggest treatment when additional studies will be available.Well designed randomized controlled trials are needed in order to understand the impact of different types of anti-PL on implantation and consequently on infertility, before using them in everyday clinical management of infertile women with recurrent ET failures. Moreover, cost-effectiveness studies should be conducted to evaluate benefits and costs of this approach. Well designed interventional studies might confirm presence of anti-PL antibodies as predictive markers of implantation failure, but also target them pharmacologically in women suffering from infertility or subfertility. The present meta-analysis highlights the importance of the presence of anti-β2GPI and anti-PS antibodies regarding the risk for implantation failure. Thus, it would be useful to insist on measuring them in cases of infertility at least concurrently with the more frequently measured aPL antibodies.(DOCX)Click here for additional data file.
PRISMA 2009 checklist.
(DOC)Click here for additional data file.11 May 2021PONE-D-21-07054Presence of antiphospholipid antibodies is associated with increased implantation failure following in vitro fertilization technique and embryo transfer: A systematic review and meta-analysis.PLOS ONEDear Dr. Mastorakos,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.The review deals with an interesting topic and adequately follows the reference methodological checklist.In addition to the referees' comments which deserve careful consideration, I have the following observations:- I suggest elaborating figure 1 in detail according to the Prisma 2020 version.- The tables are not immediately understandable and have very long titles. I suggest changing them with a shorter title, more immediate and clear content and adding any footnotes.- in forest plots it is necessary to modify the "success IVF" outcome with a more informative title (pregnancy rate? Delivery rate? ...)- when the I2 index is quite high, for example> 30%, it may be advisable to also provide the results using the random effect model, or to discuss why only the fixed effect model was used.Please submit your revised manuscript by Jun 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Title:- Concise and soundAbstract:- Purpose: Can you please make it more clear that you are comparing aPL between patients having IVF-ET failures and controls. No need to mention implantation rate.- Methods: Clear- Results: Clear- Conclusion: Please highlight the important finding that aB2GPI and aPS are the most associated with implantation failures.Keywords:- Please decrease the number, no need to mention the types of aPLIntroduction:- Line 57: Reference 4 should be replaced with a specific ObGyn reference- Line 62: Please add a reference- Line 63: Replace the word biochemical with serological- Line 67 to 69: I would suggest to remove because these non-criteria aPL are gaining attention in the current research- Line 70: Why are you defining aPL again, this should happen previously- Line 75: Please add a reference- Lines 75- 77: Please add little bit more data about the pathophysiology, although you have elaborated precisely in the discussion partMaterials and Methods:- Line 92: Screening by one author is a limitation. Please explain.- Line 100: The past medical history needs elaboration. Any woman with previous diagnosis of APS or thrombosis?- Line 123: Here, you mentioned that you will be assessing non-criteria aPL which means that they have clinical significance contrary to what you have preciously mentioned in the introduction.- Line 149: Can you please submit a clearer version of figure 1, I can barely see it.- Line 156: Can you please elaborate about the prospective nature of the studies (Trial, cohort, etc...)- Line 160: After how many days did the studies assess for hCG?- Line 181: I would suggest mentioning the correct references for the cut-offs and manufacturer.Results:- Please explain what you mean by population index, which will make it easier for the general reader.- Lines 235-239: Here, you compare the isotypes of aCL. What is intention? Later on, you completely neglect the significant findings.Discussion:- Lines 279- 282: Please make shorter.- Lines 282-287: How did the data differ between retrospective and prospective studies? Could be this a limitation?- Lines 294-301: Here, you are discussing non-criteria aPL. Were these confirmed in any of the studies that you have used? Some non-criteria aPL are still considered acute-phase reactants.- Line 304: Would suggest using better references. For example, Rodrigues VO, Soligo AGES, Pannain GD. Antiphospholipid Antibody Syndrome and Infertility. Rev Bras Ginecol Obstet. 2019 Oct;41(10):621-627. English. doi: 10.1055/s-0039-1697982. Epub 2019 Oct 28. PMID: 31658490. and El Hasbani G, Khamashta M, Uthman I. Antiphospholipid syndrome and infertility. Lupus. 2020 Feb;29(2):105-117. doi: 10.1177/0961203319893763. Epub 2019 Dec 12. PMID: 31829084.- Lines 304- 310: We already know that APS can cause early miscarriage. This is out of the scope of the systematic review.- Line 319: Reference please- Line 324: Reference please- Lines 327- 332: What about other non-criteria aPL? Do we have preliminary data on their relationship with implantation failure?- The discussion would benefit more from highlighting the important finding of the role of aB2GPI and aPS in implantation failure.Figures:- Would suggest making them more clear to the general reader. What do you mean by panel A, B, etc... Please elaborate and guide us through every figure.References:References 21, 25, 30, and 38 need proper citations. There are errors in these references.Reviewer #2: This is an interesting study and the authors have collected a unique dataset. The paper is generally well written and structured. May be only a few remarks:1) 62 line - "Clinical manifestations of APS include fertility difficulties and pregnancy complications such as repeated miscarriages".- Please add thrombosis as a one of the main manifestations of APS (besides obstetric complications)2) 64-65 line - "When circulating anti-PL antibodies are positive on two occasions six weeks to six months apart, the diagnosis of APS is confirmed".- In fact, there are no data to validate this interval but updated APS criteria increased the interval from 6 to 12 weeks. So, officially this interval consists of 12 weeks.Overall, this is a clear, concise, and well-written manuscript that deserved to be accepted and published.********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: NoReviewer #2: Yes: Khizroeva[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.28 Oct 2021George Mastorakos, MD, DScProfessor of EndocrinologyUniversity of AthensGreece3, Neofytou Vamva st10674 AthensGreeceFax: 0030-210-3636229e-mail: mastorakg@gmail.comAlessio Paffoni, PhDAcademic EditorPLOS ONEJuly 30th , 2021,Dear Dr Alessio Paffoni,We have received your revision letter of our paper entitled: "Presence of antiphospholipid antibodies is associated with increased implantation failure following in vitro fertilization technique and embryo transfer: A systematic review and meta-analysis." (PONE-D-21-07054). We thank you and the reviewers for your valuable time and appreciate the comments to which we respond as follows.Editor’s comments:Comment 1. I suggest elaborating figure 1 in detail according to the Prisma 2020 version.Response to comment 1: We thank the editor for this useful suggestion. We have now elaborated figure 1 according to the Prisma 2020 version.Comment 2. The tables are not immediately understandable and have very long titles. I suggest changing them with a shorter title, more immediate and clear content and adding any footnotes.Response to comment 2: We thank the editor for this remark. We have shortened the titles and added an explanatory footnote at each table.Comment 3. In forest plots it is necessary to modify the "success IVF" outcome with a more informative title (pregnancy rate? Delivery rate? ...)Response to comment 3: We thank the editor for the astute remark. We have now modified all the forest plots to show the risk for implantation failure in relation to the presence of antiphospholipid antibodies (+Abs). The term “success IVF” has been removed from the forest-plots and specific explanations are now included in the legends of the Figures.Comment 4. When the I2 index is quite high, for example> 30%, it may be advisable to also provide the results using the random effect model, or to discuss why only the fixed effect model was used.Response to comment 4: We thank the Editor for this comment. In line with guidance by Cochrane, in all provided results the random effect model has been employed. This is clearly stated in the Material and methods section (page 6, line 140-141): “… For data synthesis the random effect model was employed as significant clinical and methodological heterogeneity among the included studies was anticipated. …”.Reviewers’ commentsReviewer #1Title:- Concise and soundResponse: We thank the reviewer for stating that the title of our manuscript was “concise and sound”.Abstract:- Purpose: Can you please make it more clear that you are comparing aPL between patients having IVF-ET failures and controls. No need to mention implantation rate.Response: We thank the reviewer for this remark. We have now rephrased the aim of the study to make it more clear that presence of aPL is compared between patients having IVF-ET failures and controls. It states now in the Abstract section (page 2, line 25-30): “…A systematic review and meta-analysis was conducted comparing the presence of anti-phospholipid (anti-PL) antibodies between women of reproductive age, without diagnosis of antiphospholipid syndrome, who experienced at least two implantation failures following in vitro fertilization and embryo transfer (IVF-ET), and either women who had a successful implantation after IVF-ET or women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET. …”- Methods: Clear- Results: ClearResponse: We thank the reviewer for stating that the methods and the results in the abstract section were clear.- Conclusion: Please highlight the important finding that aB2GPI and aPS are the most associated with implantation failures.Response: We thank the reviewer for this suggestion. We have now included the following comment in the Abstract section (page 2, line 44-45): “…The prevalence of antiphospholipid antibodies, particularly that of anti-beta2 glycoprotein-I and anti-phosphatidylserine antibodies, in women experiencing at least two implantation failures in IVF-ET…”.Keywords:- Please decrease the number, no need to mention the types of aPLResponse: We have now decreased the number of keywords, as follows: “Antiphospholipid, anti-phosphatidylserine, anti-beta2 glycoprotein I, implantation, IVF”.Introduction:- Line 57: Reference 4 should be replaced with a specific ObGyn referencehttps://obgynkey.com/chapter-20-single-embryo-transfer/Response: We thank the reviewer for this suggesion. As suggested reference 4 has now been replaced by: Martine Nijs, Single Embryo Transfer in Stewart, J. (Ed.). (2019). Subfertility, Reproductive Endocrinology and Assisted Reproduction. Cambridge: Cambridge University Press. doi:10.1017/9781316488294- Line 62: Please add a referenceResponse: The following reference is now added: Miyakis S, Lockshin MD, Atsumi T, Tincani A, Vlachoyiannopoulos PG, Krilis SA et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J. Thromb Haemost. 2006 Feb; 4(2):295-306. doi: 10.1111/j.1538-7836.2006.01753.x- Line 63: Replace the word biochemical with serologicalResponse: We thank the reviewer for this suggestion. The word “biochemical” has now been replaced (page 3, line 65) with the word “serological”.-Line 67 to 69: I would suggest to remove because these non-criteria aPL are gaining attention in the current researchResponse: We thank the reviewer for this suggestion. Thus, the sentence (previously page 4, line 67-69): “… Other types of anti-PL antibodies have been employed in the past and still found in older literature although not included any more in the APS definition by international consensus committees. …” has been removed.- Line 70: Why are you defining aPL again, this should happen previouslyResponse: We thank the reviewer for this suggestion. Accordingly, we have moved the comment: “…Antiphospholipid antibodies represent a heterogeneous group of antibodies, which recognize various phospholipids, phospholipid-binding proteins, and phospholipid protein complexes. …” from previously line 70 to line 61-63.- Line 75: Please add a referenceResponse: The following reference is now added: Carp HJ, Meroni PL, Shoenfeld Y. Autoantibodies as predictors doi: 10.1093/rheumatology/ken154- Lines 75- 77: Please add little bit more data about the pathophysiology, although you have elaborated precisely in the discussion partResponse: We thank the reviewer for this suggestion. We have now added little more data to be more explanatory but not repetitive. It states now as follows (page 3, line 72-76): “…Antiphospholipid antibodies, especially anti-beta2 glycoprotein I (anti-β2GPI) antibodies, in pregnancy, appear to act directly on trophoblasts by activating pro-apoptotic and pro-inflammatory mechanisms6. At the same time, thrombosis of placental chorionic arteries and activation of the complement system intravascularly lead to the cell death of the trophoblast by decreasing trophoblast viability, syncytialization, and capacity for invasion. ....”.Materials and Methods:- Line 92: Screening by one author is a limitation. Please explain.Response: We thank the reviewer for this remark. Literature was screened systematically by one author (EP) (as already mentioned in the manuscript) following the “Preferred reporting Items for Systematic Reviews and Meta-Analyses and detailed search strategy”which is available in the online supplement. Search strategy was validated by G.M. Wherever a discrepancy was raised by E.P., G.M. was consulted. This information is now added in the Materials and methods section (page 4, line 94-95): “…Search strategy was validated by GM. When EP raised a discrepancy, GM was consulted. …”.-Line 100: The past medical history needs elaboration. Any woman with previous diagnosis of APS or thrombosis?Response: We thank the reviewer for raising this point. There was not any woman with previous diagnosis of APS or thrombosis. This it is stated in the inclusion criteria (page 5, line 108-109): “… All study populations should be consisted by healthy women of reproductive age not suffering from any known autoimmune, endocrine or infectious diseases. …” and it was followed accordingly. This piece of information is now included in the Material and methods section (page 4, line 100-101): “…past medical history of women included in each study was retrieved (no women suffered from APS or had a history of thrombosis)…”.- Line 123: Here, you mentioned that you will be assessing non-criteria aPL which means that they have clinical significance contrary to what you have previously mentioned in the introduction.Response: We thank the reviewer for this astute remark. In the introduction section is stated: “According to revised Sapporo criteria, diagnosis of APS takes into account lupus anticoagulant (LA), anti-cardiolipin (anti-CL) antibodies or anti-β2glycoprotein I (anti-β2GP I) antibodies of either IgG or IgM isotype. Other types of anti-PL antibodies have been employed in the past and still found in older literature although not included any more in the APS definition by international consensus committees.” However in the present meta-analysis we aimed at comparing presence of aPL in women of reproductive age without diagnosis of antiphospholipid syndrome, who experienced implantation failure following in vitro fertilization and embryo transfer (IVF-ET) and in women who had a successful implantation after IVF-ET or in women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (please see response to Comment for abstract/purpose of the study of Reviewer #1). Thus, we opted to include comparisons of aPL included in the Sapporo criteria for APS diagnosis (anti-CL; LA; anti-β2GPI antibodies;) as well as of six more aPL (anti-PS; anti-PC; anti-PE; anti-PI; anti-PG; anti-PA antibodies) which gained importance in recent literature for APS diagnosis. This point is now clarified in the Material and methods section (page 5, line 119-124): “…Secondary outcomes extracted from the selected studies were presence or not of: anticardiolipin (anti-CL), lupus anticoagulant (LA) and anti-β2GPI antibodies (all three representing aPL included in the Sapporo criteria for APS diagnosis), as well as anti-phosphatidylserine (anti-PS), anti-phosphatidylcholine (anti-PC), anti-phosphatidylethanolamin (anti-PE), anti-phosphatidylinositol (anti-PI), anti-phosphatidylglycerol (anti-PG) and anti-phosphatidic acid (anti-PA) antibodies which have gained importance in recent literature for APS diagnosis. …”.- Line 149: Can you please submit a clearer version of figure 1, I can barely see it.Response: We thank the reviewer for this comment. Figure 1 was modified according the journal’s requirement and now a new version has been submitted.- Line 156: Can you please elaborate about the prospective nature of the studies (Trial, cohort, etc...)Response: We thank the reviewer for this remark. We have now clarified in Results section (page 7, line 155-156): ‘‘…(ten and seven studies were retrospective and prospective cohort studies, respectively)) …’’.- Line 160: After how many days did the studies assess for hCG?Response: Unfortunately, studies did not report after how many days hCG was determined. This is now clarified in the Results section (page 7, line 161-162): “…in none of the studies the timing of hCG measurement was reported …”.- Line 181: I would suggest mentioning the correct references for the cut-offs and manufacturer.Response: We thank the Reviewer for raising this point.This information is now included in the material and methods section (page 8, line 181-192): “…Diagnostic cut-offs for the antibodies were reported in: Qublan H. et al (anti-CL antibodies positive >10 IU/ml; qualitative positivity or negativity for LA)13; Vaquero E et al (anti-CL antibodies positive >20; qualitative positivity or negativity for LA)18; Bellver et al (anti-CL antibodies positive >20 gPL/ml or mPL/ml for IgG or IgM isotype, respectively; qualitative positivity or negativity for LA)21; Sanmarco M. et al [antibodies positive for anti-CL: IgG ≥20 GPLU; for anti-β2GPI IgG ≥10 B2GU; for aPE IgG ≥15 PEGU (GPLU, MPLU, B2GU and PEGU are arbitrary units for optical density)] 20 ; Geva E. et al (anti-CL antibodies positive >23 GPLU) 14.In six studies positivity was based on optical density measurement exceeding the 99th or the 95th percentile of measurements established for each phospholipid in healthy individuals of reproductive age 11, 14, 17, 19, 22, 23. Six studies did not report diagnostic cut-offs 3, 9, 10, 15, 16, 24. Manufacturers of the assays are reported in all studies except two 13, 20. …”.Manufacturers of the assays are reported in all studies except two (14, 21). This information is now reported in detail in the online Appendix in the characteristics of each study.Results:- Please explain what you mean by population index, which will make it easier for the general reader.Response: We thank the reviewer for this comment. We have now added in the Materials and Methods section the following clarification (page 4, line 93-94): “…The term population index refers to the total number of women who experienced at least two implantation failures after IVF-ET. …”.- Lines 235-239: Here, you compare the isotypes of aCL. What is intention? Later on, you completely neglect the significant findings.Response: We thank the reviewer for this remark. Three studies out of six in subgroup B point out two distinct aCL isotypes (aCL/Ig-G, aCL/Ig-M) in their reporting of aCL evaluation. For the sake of detailed reporting we decided to mention this information. However, meta-analysis was conducted only for the aCL/Ig-G isotype because, for this isotype, a non-significant heterogeneity among the selected studies was observed, whereas for the aCL/Ig-M isotype the heterogeneity observed among the selected studies was substantial. Thus, in line with the methodology and guidance of Cochrane handbook a meta-analysis was not meaningful for the aCL/Ig-M isotype. This information is now added in the Results section (page 11 line 245-250). “...Three studies out of six in subgroup B point out two distinct aCL isotypes (aCL/Ig-G, aCL/Ig-M) in their reporting of aCL evaluation. However, meta-analysis was conducted only for the aCL/Ig-G isotype because, for this isotype, a non-significant heterogeneity among the selected studies was observed, whereas for the aCL/Ig-M isotype the heterogeneity observed was substantial. Thus, in line with the methodology and guidance of Cochrane handbook, a meta-analysis was not meaningful for the aCL/Ig-M isotype. …”.Discussion:- Lines 279- 282: Please make shorter.Response: We thank the reviewer for this recommendation. We have now made it shorter, as follows (page 12, line 295-297): “…This meta-analysis looked into multiple anti-PL antibodies, including newer markers (i.e. anti-PS antibodies) aiming at reporting a quantitative result based on the homogeneity and similarity in the findings of the included studies. …”.-Lines 282-287: How did the data differ between retrospective and prospective studies? Could be this a limitation?Response: We thank the reviewer for raising this point. The aim of this systematic review was to evaluate whether the presence of antiphospholipid antibodies has an impact on implantation rate. The retrospective studies compared the presence of antiphospholipid antibodies as a risk factor between women who experienced implantation failure after IVF-ET and controls, extracting this information from the medical history and previous laboratory. The prospective studies evaluate the presence of antiphospholipid antibodies between the women who experienced implantation failure after IVF-ET and controls, extracting this information after the enrollment of individuals it the study. The compared groups (population index and controls) are well defined in both cases. Thus, the accuracy and reliability of the extracted results cannot be limited in either case. This information is now added in the Discussion section (page 13-14 line 297-299): “...Both in the retrospective and prospective studies included in the present meta-analysis the compared population indices and control groups are well defined. Thus, the accuracy and reliability of the extracted results cannot be limited in either case. …”.- Lines 294-301: Here, you are discussing non-criteria aPL. Were these confirmed in any of the studies that you have used? Some non-criteria aPL are still considered acute-phase reactants.Response: We thank the reviewer for this comment. Indeed, as previously clarified (please see our response to the Comment for Line 123 of the Material and Methods section of this Reviewer), in this meta-analysis, were included not only studies which evaluated aPLs of the revised Sapporo criteria (LA, anti-CL, anti-β2GP I) but also types of anti-PL (anti-PS, anti-PC, anti-PE, anti-PI, anti-PG, anti-PA) antibodies which have gained importance in recent literature for APS diagnosis. Thus, it should be investigated whether these antibodies, not routinely measured, are involved as well in pathophysiologic aspects of implantation. To clarify this point, we rephrased as follows (page 13, line 317-322): “…Of note, in this meta-analysis, were included not only studies reporting anti-PL antibodies included in the revised Sapporo criteria (LA, anti-CL, anti-β2GP I) but also anti-PL antibodies (anti-PS, anti-PC, anti-PE, anti-PI, anti-PG, anti-PA) which have gained importance for APS diagnosis in recent literature. Thus, it should be investigated whether these antibodies, not routinely measured, are involved as well in pathophysiologic aspects of implantation. …”.- Line 304: Would suggest using better references. For example, Rodrigues VO, Soligo AGES, Pannain GD. Antiphospholipid Antibody Syndrome and Infertility. Rev Bras Ginecol Obstet. 2019 Oct;41(10):621-627. English. doi: 10.1055/s-0039-1697982. Epub 2019 Oct 28. PMID: 31658490. and El Hasbani G, Khamashta M, Uthman I. Antiphospholipid syndrome and infertility. Lupus. 2020 Feb;29(2):105-117. doi: 10.1177/0961203319893763. Epub 2019 Dec 12. PMID: 31829084.Response: We thank the reviewer for the suggestion. We have now replaced the references.- Lines 304- 310: We already know that APS can cause early miscarriage. This is out of the scope of the systematic review.Response: We thank the reviewer for suggesting this accurate improvement of our manuscript. We have now removed any reference to APS alone, although we kept references reporting data from presence of either APL or APS in the same study. Thus, the following sentence is now removed (previous page 14, line 307-310): “…In a 10-year follow-up of an observational, prospective study of 1000 patients (82% women) with primary or secondary APS, 127 (15.5%) women became pregnant (188 pregnancies) with 137 (72.9%) of them ending with healthy term neonate. The reported complications included early pregnancy losses (16.5% of all pregnancies) 34….”.- Line 319: Reference pleaseResponse: The following reference is now added: Nodler J et al. Elevated antiphospholipid antibody titers and adverse pregnancy outcomes: analysis of a population-based hospital dataset. BMC Pregnancy Childbirth. 2009 Mar 16;9:11 doi: 10.1186/1471-2393-9-11- Line 324: Reference pleaseResponse: The following references are now added:Marchetti et al. Obstetrical Antiphospholipid Syndrome: From the Pathogenesis to the Clinical and Therapeutic Implications. Clinical and Developmental Immunology Volume 2013 DOI: 10.1155/2013/159124Arakawa M, Takakuwa K, Honda K, Tamura M, Kurabayashi T, Tanaka K. Suppressive effect of anticardiolipin antibody on the proliferation of human umbilical vein endothelial cells. Fertil Steril. 1999 Jun;71(6):1103-7. doi: 10.1016/s0015-0282(99)00117-x. PMID: 10360918- Lines 327- 332: What about other non-criteria aPL? Do we have preliminary data on their relationship with implantation failure?Response: In this section of the Discussion we report data which associate aPLs of the revised Sapporo criteria (LA, anti-CL, anti-β2GP I) with implantation failure . From the other types of anti-PL (anti-PS, anti-PC, anti-PE, anti-PI, anti-PG, anti-PA) antibodies, only anti-PS have been associated, to our knowledge, directly with implantation failure as reported in this section. To clarify this point we have now added the following (page 14, line 351-352): “…To our knowledge, the presence of anti-PC, anti-PE, anti-PI, anti-PG, or anti-PA antibodies has not as yet been associated directly with implantation failure. …”.- The discussion would benefit more from highlighting the important finding of the role of aB2GPI and aPS in implantation failure.Response: We thank the reviewer for stating that the findings of the role of anti-β2GPI and anti-PS are important. We have now highlighted these findings, as follows (page 15, line 371-374): “…The present meta-analysis highlights the importance of the presence of anti-β2GPI and anti-PS antibodies regarding the risk for implantation failure. Thus, it would be useful to insist on measuring them in cases of infertility at least concurrently with the more frequently measured aPL antibodies.…’’Figures:- Would suggest making them more clear to the general reader. What do you mean by panel A, B, etc... Please elaborate and guide us through every figure.Response: We thank the reviewer for the comment. Legends to Figures 2 and 3 have been rephrased to explain more accurately the content of the represented Panels in each Figure as follows:Figure 2: Meta-analyses assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup A between women with at least two implantation failures in IVF-ET (population index) vs. women with one successful IVF-ET (control). Panel A: meta-analysis assessing the risk for implantation failure in relation to the presence or not of any type of anti-PL antibodies. Panel B: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL antibodies. Panel C: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies. +Abs: positive antibodiesFigure 3: Meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PL antibodies in the studies in subgroup B between women with at least two implantation failures in IVF-ET (population index) vs women with at least one successful spontaneous pregnancy or unselected healthy fertile women with no history of IVF-ET (control). Panel A: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-CL-IgG antibodies. Panel B: meta-analysis assessing the risk for implantation failure in relation to the presence or not of LA antibodies. Panel C: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti- β2GPI antibodies. Panel D: meta-analysis assessing the risk for implantation failure in relation to the presence or not of anti-PS antibodies. +Abs: positive antibodiesReferences:-References 21, 25, 30, and 38 need proper citations. There are errors in these references.Response: The previous references 21, 25 and 38 have now been corrected.Reviewer #2:This is an interesting study and the authors have collected a unique dataset. The paper is generally well written and structured. May be only a few remarks:We thank the reviewer for stating that: “…This is an interesting study and the authors have collected a unique dataset. The paper is generally well written and structured. …”.-1) 62 line - "Clinical manifestations of APS include fertility difficulties and pregnancy complications such as repeated miscarriages".- Please add thrombosis as a one of the main manifestations of APS (besides obstetric complications)Response: We thank the reviewer for the comment. We have now rephrased this point in the Introduction section (page 3, line 63-64): “…Clinical manifestations of APS include fertility problems and pregnancy complications (such as repeated miscarriages) as well as venous or arterial thrombosis. …”.-2) 64-65 line "When circulating anti-PL antibodies are positive on two occasions six weeks to six months apart, the diagnosis of APS is confirmed".- In fact, there are no data to validate this interval but updated APS criteria increased the interval from 6 to 12 weeks. So, officially this interval consists of 12 weeks.Response: We thank the reviewer for this remark. We have now clarified this in the Iintroduction section (page 3, line 66-67): “…When circulating anti-PL antibodies are positive at initial diagnosis, testing should be repeated at least 12 weeks later to confirm diagnosis of APS. …”.We have responded to all of the Reviewers’ comments. We believe, and hope that you will agree as well, that we have addressed satisfactorily all points raised so that our paper will be found adequate for publication in your prestigious Journal.With best regardsGeorge Mastorakos, MD, DScProfessor of EndocrinologyUniversity of Athens Medical Schoole-mail: mastorakg@gmail.com(On behalf of the authors)Submitted filename: response to reviewers.docClick here for additional data file.17 Nov 2021Presence of antiphospholipid antibodies is associated with increased implantation failure following in vitro fertilization technique and embryo transfer: A systematic review and meta-analysis.PONE-D-21-07054R1Dear Dr. Mastorakos,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.Kind regards,Alessio Paffoni, PhDAcademic EditorPLOS ONEAdditional Editor Comments (optional):My opinion is that the authors responded comprehensively to the referees' comments. The manuscript in its present form is satisfactory and meets the publication criteriaReviewers' comments:15 Dec 2021PONE-D-21-07054R1Presence of antiphospholipid antibodies is associated with increased implantation failure following in vitro fertilization technique and embryo transfer: A systematic review and meta-analysis.Dear Dr. Mastorakos:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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Authors: E Pierro; G Cirino; M R Bucci; N Lazzarin; C L Andreani; S Mancuso; A Lanzone; P Navarra Journal: Fertil Steril Date: 1999-02 Impact factor: 7.329
Authors: José Bellver; Sergio R Soares; Claudio Alvarez; Elkin Muñoz; Alberto Ramírez; Carmen Rubio; Vicente Serra; José Remohí; Antonio Pellicer Journal: Hum Reprod Date: 2007-12-10 Impact factor: 6.918
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