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Ovarian reserve in women with primary antiphospholipid syndrome LYS Yamakami1, PC Serafini1, DB de Araujo2,3, E Bonfa´2, EP Leon2, EC Baracat1 and CA Silva2,4 1Department of Gynecology; 2Division of Rheumatology; 3Department of Rheumatology, Hospital do Servidor Pu´blico Estadual de Sa˜o Paulo, Sa˜o Paulo, Brazil; and 4Pediatric Rheumatology Unit, Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil Objective: The objective of this paper is to evaluate ovarian reserve in primary antiphospho-lipid syndrome (PAPS) women and the association between ovarian reserve tests and clinicaland laboratorial parameters, and anti-corpus luteum antibody (anti-CoL). Methods: Wescreened 85 female patients between 18 to 40 years old with APS. Of these, 67 patients wereexcluded because of association with other autoimmune diseases (n ¼ 42), contraindication orunwillingness to stop hormonal contraceptive (n ¼ 21), current pregnancy or breastfeeding(n ¼ 3) and previous ovarian surgery (n ¼ 1). Therefore, a cross-sectional study was conductedin 18 PAPS patients and 24 healthy women. They were evaluated at early follicular phase withmeasurement of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, andanti-Mu¨llerian hormone (AMH) and sonographic antral follicle count (AFC). Serum meas-urement of anti-CoL was determined by immunoblot analysis. All analyses were performedafter at least six months from the last intake of hormonal contraceptive and resumption ofmenstruation. Results: The mean age was comparable in PAPS and controls (33.0  5.0 vs.
30.4  7.0 years; p ¼ 0.19). Regarding ovarian reserve tests, the frequencies of low AFC (10)(56% vs. 22%, p ¼ 0.04) and very low AFC (5) (37% vs. 9%, p ¼ 0.04) were significantlyhigher in PAPS patients than controls. Trends of higher frequencies of reduced (<1.0 ng/ml),low patients (p ¼ 0.08, p ¼ 0.07 and p ¼ 0.07, respectively). FSH, LH and estradiol were similarin patients and controls. There was no association between low ovarian reserve and specifictypes of antiphospholipid antibodies. Anti-CoL was solely observed in PAPS patients (11%vs. 0%; p ¼ 0.177) and was not related to ovarian reserve tests. Conclusion: Women sufferingfrom PAPS possessed reduced ovarian reserve, with prevalence greater than 50%.
(2014) 23, 862–867.
Key words: Antiphospholipid syndrome; autoimmune diseases; anti-Mu¨llerian hormone;antral follicle count; ovarian reserve test they might have additional burdens on their repro-ductive health.1,2 Antiphospholipid syndrome (APS) is an auto- Likewise, it is known that aging, smoking and ovarian surgery decrease the quantity and quality either as an isolated condition known as primary of primordial follicles in the ovaries and, ultim- APS (PAPS) or in association with other auto- ately, the ovarian reserve (OR).3,4 Conditions immune diseases, particularly systemic lupus ery- such as autoimmune oophoritis,5,6 ovarian ische- thematosus (SLE).1 Females afflicted by PAPS mia7,8 and corpus luteum hemorrhage9 may lead have an increased risk of complications during to diminished OR. Females suffering from PAPS pregnancy, and studies have also implied that are susceptible to the above-mentioned complica-tions, yet the medical literature lacks informationon how this relevant autoimmune disease affectsthe OR of these women.
Correspondence to: Clovis Artur Silva, Faculdade de Medicina da Accordingly, we assessed the OR profile of USP, Reumatologia, Av Dr Arnaldo, 455, 3 andar, sala 3105, Sa˜o women suffering from PAPS by performing hormo- Paulo – SP – 01246-903, Brazil.
nal evaluations and state-of-the-art pelvic ultra- Email: [email protected] and CAS should be considered similar in author order.
sound scanning, and we compared these results Received 2 November 2013; accepted 6 March 2014 with the OR profile of healthy women.
! The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav Ovarian reserve in PAPSLYS Yamakami et al.
Materials and methods Demographic data, PAPS manifestations, andtreatment Patients and controls Each patient's medical records were carefully Eighty-five patients aged 18 to 40 years old with reviewed concerning clinical, immunological and treatment findings. PAPS manifestations were Outpatient Clinic of the Rheumatology Division defined as vascular thrombosis (one or more clin- ical episodes of arterial, venous, or small-vessel Medicina da Universidade de Sa˜o Paulo and thrombosis in any tissue or organ) or pregnancy were invited to participate in this study. These morbidity (fetal death; premature birth before the patients were screened for PAPS during the 34th week of gestation because of eclampsia, period from April 2010 to March 2013. All severe pre-eclampsia, or placental insufficiency; patients had a previous history of thrombosis, or three or more consecutive spontaneous abor- and none were newly diagnosed. None had throm- tions).1 All patients were on long-term warfarin bosis at entry and all were on long-term use of treatment. Brazilian socio-economic stratification warfarin. The median time of warfarin use was was carried out according to the Associac¸a˜o 5.05 years (0.7–12.9). Participants were excluded from the study if they were pregnant, breastfeed- Mercados criteria.10 Body mass index (BMI) was ing, unwilling to participate in the study, had med- defined as the weight in kilograms/height in ical contraindications, possessed an increased risk meters2 (kg/m2).
of corpus luteum bleeding after stopping use ofprogestogen-only contraceptives,9 underwent pre- Gynecologic evaluation vious ovarian surgery, required systemic chemo- Complete history and physical examination includ- therapy, underwent pelvic radiotherapy, were ing history of the age at menarche and detailed currently using a long-acting gonadotropin release obstetric data were recorded.
hormone agonist (GnRH-a), presented with anadditional autoimmune disease, or had an end- stage renal disease. None of them were underestrogen contraceptive during the study or any- Blood samples were drawn by venipuncture in the time during their treatment.
early follicular phase (menstrual cycle day 2 Of the 85 women, 67 were excluded because of through 4). All sera were processed in a centrifuge an associated autoimmune condition (n ¼ 42), and stored at 70C until analysis. Follicle-stimu- contraindications or unwillingness to stop use of hormonal contraceptives (n ¼ 21), currently being 12.5 IU/l), luteinizing hormone (LH) (reference pregnant or breastfeeding (n ¼ 3), or because of level: 2.4–12.6 IU/l) and estradiol (E2) (reference previous ovarian surgery (n ¼ 1). Therefore, the level: 166 pg/ml) were measured by radioimmuno- assay using a commercial kit (CobasÕ, Roche, women with PAPS. The control group included Mannheim, Germany). Intra- and inter-assay coef- ficients of variation were recommended by the recruited from the primary care clinic nearby our manufacturer and were limited to 5.7 and 3.6%, tertiary hospital. The control volunteers were sub- respectively. FSH concentrations were considered ject to the same inclusion and exclusion criteria.
elevated when the levels were equal to or greater All PAPS participants voluntarily agreed to dis- than 10 IU/l.4,11,12 continue the use of hormonal contraceptives Anti-Mu¨llerian hormone (AMH) was measured during a period of at least six months and had by enzyme-linked immunosorbent assay (ELISA) at least two consecutive menstruations to reveal in duplicated samples (AMH Gen II ELISA, the onset of normal endocrinological hypothala- Beckman Coulter Inc, Brea, CA, USA). Intra- mic-pituitary-ovarian axis activity. None of our and interassay coefficients of variation were limited PAPS patients and controls had hypergonado- to 5.4% and 5.6%, respectively. AMH levels were trophic amenorrhea, which would suggest primary classified by cutoff values: lower than 1.0 ng/ml ovarian insufficiency (high levels of FSH >40 IU/l (reduced), lower than 0.5 ng/ml (low) and lower and sustained amenorrhea).9 The local ethics com- than 0.2 ng/ml (negligible).13,14 mittee of our university hospital approved this Transvaginal ultrasound was carried out to study, and informed consent was obtained from determine the antral follicle count (AFC) and all participants.
the mean ovarian volume on the same day that Ovarian reserve in PAPS LYS Yamakami et al.
the blood was drawn. Transvaginal ultrasound Statistical analysis was not performed in women not engaged in Results are shown as the mean  standard devi- ation (SD) or median (range) for continuous vari- were performed by an experienced, trained, repro- ables and as percentages for categorical variables.
ductive specialist (LYSY) using a 6.5 MHz endo- For continuous variables, data were compared by vaginal transducer (HD3, Philips Ultrasound, the t test and Mann-Whitney test to evaluate dif- Bothell, WA, USA) who was blinded to the vol- ferences between the PAPS group and control unteers' diagnoses and blood test results. Ovaries group. For categorical variables, differences were were scanned in axial and longitudinal planes, and assessed by Fisher's exact test. The level of signifi- at least two measurements of length (L), width cance was set at 5% (p < 0.05).
(W) and thickness (T) were obtained and used tocalculate mean ovarian volume using the formulafor an ellipsoid (L  W  T  0.523). The AFCwas calculated by measuring follicles with sizes from 2 to 10 mm in both ovaries.4,15 AFC meas-urements were classified clinically as: normal when Histories of arterial thrombosis, venous thrombosis there were 10 follicles, low when there were 10 and pregnancy morbidity were detected in 17%, but >5 follicles, and very low when there were 5 83%, and 33% of the PAPS patients, respectively.
LA, aCL antibodies, and anti-b2-GPI antibodieswere detected in 67%, 83% and 11% of the PAPS Detection of anti-corpus luteum (anti-CoL) and patients, respectively. All PAPS patients were nega- other autoantibodies tive for anti-dsDNA, anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B autoantibodies.
The presence of autoantibodies directed toward a The demographic features, gynecologic history, 67 kDa protein in the corpus luteum was deter- OR test results, and anti-CoL statuses of the PAPS mined in the patient and control samples using patients and controls are depicted in Table 1. The mean age was similar between the PAPS group and Briefly, crude tissue and cell extracts obtained controls (33.0  5.0 vs. 30.4  7.0 years; p ¼ 0.189).
from bovine corpus luteum (100 mg/well) were sub- The BMI, smoking status and the remaining demo- mitted to polyacrylamide gel electrophoresis under graphic features were similar in both groups denaturing and reducing conditions (with sodium (p > 0.05). The history of deliveries was superior dodecyl sulfate and b2-mercaptoethanol). Proteins in PAPS patients when compared to controls (1 were then electrophoretically transferred to a nitro- (0–6) vs. 0 (0–2), p ¼ 0.02), and there were fewer cellulose membrane. The membrane strips were fur- pregnancies per woman in the PAPS group than ther incubated with blocking buffer (5% skimmed in the controls (17% vs. 54%, p ¼ 0.02).
milk in phosphate-buffered saline (PBS)) and The frequencies of low and very low AFC were immunoprobed by incubation with serum samples greater in PAPS patients than in controls (low diluted 1:10. Reactivity was tagged with an anti- AFC: 56% vs. 22%, p ¼ 0.042; very low AFC: human immunoglobulin (Ig)G alkaline phosphat- 37% vs. 9%, p ¼ 0.045). Reduced, low and negli- ase conjugate and visualized using appropriate gible AMH levels were observed in PAPS patients more often than in controls (p Lupus anticoagulant (LA) was detected accord- ¼ 0.08, p ¼ 0.07 and ing to the guidelines of the International Society on ¼ 0.07, respectively). Serum FSH, LH and estra- Thrombosis and Hemostasis.17 Presence of anticar- diol levels were similar in PAPS patients and con- diolipin (aCL) antibodies and anti-b2-glycoprotein trols (p > 0.05). The occurrence of anti-CoL I (anti-b2-GPI) IgG and IgM were analyzed by antibodies was observed in two PAPS patients ELISA.18 The presence of additional autoantibo- (11%) and no controls (0%) (p ¼ 0.177), and one dies, including anti-double-stranded DNA (anti- of these PAPS patients had reduced OR (Table 1).
Crithidia luciliaea substrate), anti-ribonucleopro-tein (anti-RNP), anti-Sm proteins (hemagglutin- ation assay with rabbit thymus extract), anti-Ro/SS-A and anti-La/SS-B (counterimmunoelectro- To our knowledge, this is the first study to indicate phoresis using human spleen extract as antigen) that more than half of women suffering from PAPS were detected.
have diminished OR. The main strength of this Ovarian reserve in PAPSLYS Yamakami et al.
Demographic features, gynecologic history, ovarian Follicle atresia, apoptosis, the location of primor- reserve tests, and anti-corpus luteum antibody (anti-CoL) in dial follicles near the granulosa cell layer, and ovar- primary antiphospholipid syndrome (PAPS) patients and ian blood supply could make primordial follicle cohorts more susceptible to microvascular ischemia events. Furthermore, diminished serum AMHlevels might be brought about by insults to the Demographic features ovarian microvasculature. A parallel dysfunction Current age, years could be drawn in male PAPS and SLE-related antiphospholipid patients, in whom morphofunc- tional penile alterations were observed.24,25 Diminished OR was most likely not related to Socioeconomic class C or D Gynecologic history Furthermore, unlike patients with other auto- Age at menarche, years immune diseases, our patients did not use immuno- Time between menarche and current age, years suppressive agents, such as cyclophosphamide26–29 or glucocorticosteroids,30 that could result in OR evaluation in patients with chronic auto- immune diseases may be influenced by age, throm- contraceptives, genetics and environmental factors such as smoking.26,27,29,31 Previous use of medrox- yprogesterone acetate, an option of contraception for women with positive aCL and/or LA,32 was not a relevant factor for this analysis since the drug was discontinued for at least six months and menstrual cycle had to be resumed. Additionally, patients and Mean ovarian volume, mm3a controls had comparable ages and very low tobacco smoking habits, which negates these factors as a Values expressed in mean  standard deviation, median (range) or n causes of low OR. Additionally, none of our (%). PAPS: primary antiphospholipid syndrome; BMI: body mass patients had clinical evidence of ovarian throm- index; FSH: follicle-stimulating hormone; LH: luteinizing hormone; bosis, which is in accordance with the findings AMH: anti-Mu¨llerian hormone; AFC: antral follicle count. an ¼ 16PAPS patients and 23 controls.
described for PAPS patients.33 Regarding OR evaluation, basal serum FSH, although widely available, has been demonstrated research was the evaluation of most reliable OR to have low accuracy in diagnosis of diminished markers, including AFC and serum AMH, which OR, especially at early stages, the most important were obtained at the early follicular phase to avoid phase to be identified.27 Today, AMH and AFC are intramenstrual cycle variations in AMH levels.19,20 the best non-dynamic tests to predict ovarian per- We used strict criteria to define PAPS. Moreover, formance in human reproductive treatment.26,28 all patients discontinued use of hormonal contra- AMH is produced by granulosa cells of early- ceptives for at least six months and had resumed stage follicles and has the advantage of being two or more consecutive menstrual cycles, suggest- FSH independent, which makes its level relatively ing that ovarian function was reestablished. These stable during all menstrual cycles, and it can be precautions were taken to obtain consistent OR used in early evaluation.26 AMH assessment was evaluations because it is known that AMH concen- first performed in SLE patients31 and it is con- trations decrease during hormonal contraceptive sidered a reliable ovarian marker.
use.21–23 However, the main limitation of this It is unlikely that autoimmunity influenced the study is the low sample size, which, in part, is low OR observed herein, which contrasts with our accounted for by the strict definition of PAPS and female SLE patients who showed an association the low incidence of this illness.
between anti-CoL antibodies and ovarian dysfunc- Low OR, as indicated by a significant reduction tion.16 Antibodies directed to pre-ovulatory follicle in AFC, implies that primordial follicle cohort cells and not to corpus luteum-specific antigens growth may suffer insults within an antiphospholi- may have an impact on OR in PAPS patient. This pid milieu that manifest as a reduced follicle cohort.
field of study deserves further research.
Ovarian reserve in PAPS LYS Yamakami et al.
The presence of a relatively high parity among 6 La Marca A, Brozzetti A, Sighinolfi G, Marzotti S, Volpe A, Falorni A. Primary ovarian insufficiency: Autoimmune causes.
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9 Yamakami LY, de Araujo DB, Silva CA, Baracat EC, de Carvalho was to assess OR markers in a cross-section JF. Severe hemorrhagic corpus luteum complicating anticoagula- study. A future study evaluating OR markers for tion in antiphospholipid syndrome. Lupus 2011; 20: 523–526.
prediction of pregnancy outcomes will be required.
10 Almeida PM. Crite´rio de classe econoˆmica da Associac¸a˜o Brasileira de Anunciantes (ABA) e Associac¸a˜o Brasileira dos In conclusion, the present report identifies a high Institutos de Pesquisa de Mercado (ABIPEME). In: Almeida prevalence of diminished OR in PAPS patients.
Further studies are necessary to uncover the mech- ABIPEME. ABA/ABIPEME: Sa˜o Paulo, 1991, pp.1–29.
11 Kahapola Arachchige KM, Wardrop R, Lim EM, Stuckey B, anisms by which PAPS causes ovarian impairment.
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