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The effect of parnaparin sodium on in vitro fertilization outcome: A prospective randomized controlled trial Corrado Lodigiania,⁎, Francesco Dentalib, Elena Banfia, Paola Ferrazzia, Luca Librèa, Ilaria Quagliaa, Luca Cafaroc, Emanuela Morenghid, Veronica Pacettia, Elena Zannonic, Anna Maria Baggianic, Paolo Emanuele Levi-Settic a
Humanitas Research Hospital, Cardiovascular Department, Thrombosis and Haemorragic Diseases Center, Rozzano, Milan, Italy Department of Clinical and Experimental Medicine, Insubria University, Varese, Italy c Humanitas Research Hospital, Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Rozzano, Milan, Italy. d Humanitas Research Hospital, Biostatistics Unit, Rozzano, Milan, Italy b
A R T I C L E I N F O
A B S T R A C T
Keywords: In vitro fertilization Live-birth pregnancy rate Low-molecular weight heparin Reproductive sterility Thrombophilia
Introduction: In-vitro and in-vivo models suggest the influence of low-molecular weight heparin on conception in infertile women undergoing in vitro fertilization procedures (IVF). In this randomized controlled trial we assessed whether a low-molecular weight heparin (parnaparin) could affect IVF outcomes. Materials and methods: 271 cycles were analyzed in 247 women having a first or subsequent IVF cycle at Fertility Center of Humanitas Research Hospital. Patients, without severe thrombophilia and hormonal or active untreated autoimmune disorders, were randomly allocated (1:1) to receive for the whole cycle parnaparin, or routine hormonal therapy only. The primary endpoint was the clinical pregnancy rate and the secondary endpoints included implantation rate and live birth rate. Results: The clinical pregnancy and the live birth rate were similar in treated and controls (21.5% vs. 26.7%, p = 0.389; 18.5% vs. 20.6%, p = 0.757). The abortion rate was 10.3% vs 22.9%, p = 0.319, respectively. The subgroups analysis, ≤ 35, 36–38, 39–40 years, showed the following: comparable clinical pregnancy rate (22.5% vs 38.8%, p = 0.124; 21.8% vs 17.3%, p = 0.631; 19.4% vs 23.3%, p = 0.762 respectively) and live birth rate (16.3% vs 32.7%, p = 0.099; 20.0% vs 13.5%, p = 0.443; 19.4% vs 13.3%, p = 0.731 respectively) in treated vs controls. Sensitivity analyses on women with ≥ 3 previous attempts and first enrolment only, and subgroup analyses according to trial conclusion conditioning a small sample size with low statistical power. Conclusions: Our study excludes positive effect of parnaparin, once a day for the whole cycle, on clinical pregnancy rate in infertile women undergoing in vitro fertilization techniques.
1. Introduction Since the introduction into clinical practice of in vitro fertilization procedures (IVF) [1], improvements have been made to ovarian stimulation protocols, preparation of follicles and gamete culture medium. However, the implantation rates of the pre-embryo are still low [2], which is the actual limiting factor for the success of IVF in terms of live birth rate [3]. Since embryo implantation depends on several factors, the causes of its failure, even if the embryo is transferred at blastocyst stage after full chromosomal analysis [4], remain unknown. Unfortunately, this may occasionally be a recurring phenomenon leading to despair in couples and frustration in their caregivers. Recently, some experts have advocated the use of antithrombotic drugs (heparins and aspirin) in order to improve the implantation rate [5]. ⁎
Heparin can have a positive effect in conception and early pregnancy events by altering the hemostatic response to ovarian stimulation, modulating trophoblast differentiation and invasion, and decreasing the risk of thrombosis [6]. In-vitro and in-vivo models suggest the influence of low-molecular weight heparin (LMWH) on different aspects of trophoblast adhesion and invasiveness by acting on matrix metalloproteinases and tissue inhibitors [7], cadherin-E [8,9], heparin-binding epidermal growth factor [10,11], and free insulin-like growth factor [6,12]. Clinical studies suggest an influence of heparin on IVF outcome under specific clinical conditions such as women affected by antiphospholipid antibodies (aPL) [13,14,15]. The role of heparin in patients with recurrent IVF failure without an aPL syndrome is more conflicting [16,17]. In general, original studies gave negative results. A comprehensive systematic review and meta-analysis of published literature on
Corresponding author at: Humanitas Research Hospital, Cardiovascular Department, Thrombosis and Haemorragic Diseases Center, via Manzoni 56, 20089 Rozzano, Milan, Italy. E-mail address: [email protected] (C. Lodigiani).
http://dx.doi.org/10.1016/j.thromres.2017.08.006 Received 16 June 2017; Received in revised form 7 August 2017; Accepted 13 August 2017 0049-3848/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Lodigiani, C., Thrombosis Research (2017), http://dx.doi.org/10.1016/j.thromres.2017.08.006
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a contraindication for heparin therapy were excluded. Patients with a family history (even strong) of thrombosis were not excluded from the study. According to the study protocol, patients could be enrolled in the study more than once. Participating women were submitted to a block computer-generated randomization and allocated to receive or not to receive parnaparin for the whole cycle at a daily dose of 4250 anti-Xa IU/0.4 mL and 6400 anti-Xa IU/0.6 mL, if the body weight was respectively under or over 60 kg. The randomization list was prepared by a biostatistician not involved in patient recruitment. Opaque numbered and sealed envelopes containing the allocation were managed by the study data manager, who informed the attending physicians. Data were collected on BMI, smoking habits, infertility causes, previous pregnancies, miscarriages, and IVF procedures, retrieved oocytes, transferred embryos and implantation rate, IVF outcome and parameters used for randomization.
the effect of heparin on the outcome of IVF was conducted by Seshadri et al. in 2012 which included both randomized and observational studies. The randomized studies included only small numbers of women, and had high methodological heterogeneity, and so had significant limitations. These showed no significant difference in implantation rate, clinical pregnancy, clinical miscarriage and live birth rate. Metaanalysis of the observational studies showed a significant increase in the clinical pregnancy rate and live birth rate, however the authors concluded that the potential role of heparin during IVF treatment required further evaluation in adequately powered randomized studies as they noted that the observational studies could have exaggerated the value of heparin in IVF due to selection bias. In a more recent metaanalysis, the use of LMWH significantly increased the clinical pregnancy rate and live birth rate [18,19,20]. Despite the absence of robust clinical evidence, maternal LMWH administration is used in IVF as an intervention that may improve implantation and reduce miscarriage, although clinical practice varies widely between individual clinicians and clinics. Thus, we conducted a randomized controlled trial (RCT) to assess whether LMWH administration in infertile women having a first or subsequent IVF cycle could contribute to improving the clinical outcome, with or without the presence of non-severe thrombophilia.
2.4. Laboratory tests Factor VG1691A and FIIG20210A were assessed with HTRBio-toBit-PCR Platform, Fleming Research. The activities of anticoagulant proteins were measured in human citrated plasma on IL Coagulation System® (Instrumentation Laboratory, Bedford, USA): protein C and antithrombin with an automated chromogenic assay; free protein S with automated latex ligand immunoassay; resistance to activated protein C with COATEST™ APC RESISTENCE™. Lupus anticoagulant was determined with one-stage clotting test using Simplified Dilute Russel Viper Venom (IL). Anti-Cardiolipin IgG/IgM antibodies were quantified in human serum in accordance with manufacturer's instructions (ORGENTEC, Diagnostika GmbH, Germany).
2. Materials and methods 2.1. Study design This randomized, prospective, controlled, stratified, open label and phase III study was carried out and reported according to the CONSORT (Consolidated Standards of Reporting Trials) guidelines for randomized controlled studies [21]. The study was approved by the institutional review board of our Institute and written informed consent was obtained for all women and is registered on ClinicalTrials.gov NCT02991950.
2.5. Interventions During the study period, patients underwent a controlled ovarian hyperstimulation (COH) protocol. The ovarian stimulation protocol was determined according to ovarian reserve parameters, evaluating antiMüllerian hormone (AMH), antral follicle count (AFC), and body mass index (BMI) before the treatment [22,23] and considering previous treatment cycles [24,25]. COH was performed with a GnRH agonist (Enantone die, Takeda, Italy or Triptorelin Depot 3.75 mg IM, Decapeptyl®, Ipsen, Milan, Italy) or GnRH antagonist protocol (Cetrotide®, Merck Serono, Rome, Italy; Orgalutran®, Organon, MSD-Italy) [26]. In the GnRH, agonist protocol induction was started after evidence of pituitary desensitization (absence of ovarian follicles > 10 mm and endometrial thickness < 5.4 mm on transvaginal ultrasound examination). In the GnRH antagonist protocol, women received a low dose oral contraceptive and gonadotrophins were started the first day of withdrawal bleeding [27]. The starting dose of rFSH (Puregon®, MSD-Italy; Gonal-F, Serono, Rome, Italy) or hMG (Meropur®, Ferring, Milan, Italy) was decided according to ovarian reserve parameters and or previous induction cycle for the first four days and thereafter, on the basis of transvaginal follicular parameters (number and diameter) and results of estradiol and progesterone determination, a variable dose of gonadotropin was administered until the day of ovulation trigger with a 250 mcg rhCG (Ovitrelle®, Serono, Rome, Italy Merck Serono) injection. Parnaparin sodium (Fluxum 4250 anti-Xa IU/0.4 mL or 6400 antiXa IU/0.6 mL; Alfa Wassermann S.p.A., Bologna, Italy) was administered for the whole cycle, from the day before the beginning of the stimulation phase of the cycle until the result of the procedure and, in the case of evolutive pregnancy, until delivery or the end of pregnancy. According to the study protocol, LMWH was discontinued in the case of patient refusal, toxicity or other complications precluding further therapy, or in the case of a decision by the physician. During LMWH administration, concomitant therapy with acetylsalicylic acid or steroids was not allowed, so patients on chronic treatment with these drugs
2.2. Aim of the study To assess whether a low molecular weight heparin, parnaparin, administration could contribute to improving the outcome of IVF in terms of clinical pregnancy rate in infertile women undergoing a cycle of IVF, aged ≤40 years and ≥18 years, without severe thrombophilia, and hormonal or active untreated autoimmune disorders. 2.3. Patients and methods The study was undertaken at the Fertility Center and Thrombosis and Haemorragic Diseases Center of Humanitas Research Hospital, Milan, Italy, between November 2011 to December 2015. The last participant was recruited in April 2015, and the follow-up was completed in December 2015. Originally we planned the conduct the study as a multicenter study. Subsequently it was amended as single center study due to the high management of costs. Infertile women satisfying all the inclusion and exclusion criteria were potentially eligible for the study. Women between 18 and 40 years of age who underwent a fresh IVF cycle with routine ovulation induction protocol were candidates for the study. Cycles with frozen ejaculated or testicular sperm and cycles with frozen embryos were excluded from the study, in order to include a quite homogeneous population with exclusion of all possible bias (e.g. severe male factor leading to the use of frozen or testicular sperm). Women with chronic disease (liver, renal, thyroid) or hormonal disorder not compensated with a specific therapy, with a immunological disease (e.g., autoimmune thyroiditis, connectivitis, rheumatoid arthritis, systemic lupus erythematosus), with abnormal platelet count (< 100,000/mm3), with antiphospholipid autoantibodies or other severe thrombophilia (antithrombin, protein S, protein C deficiency or homozygous FV Leiden or FIIG20210A, or double heterozygous FV Leiden and FIIG20210A), and patients with previous thrombosis or with 2
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than one IVF during the study period and were included more than once in the study. No woman was lost at follow-up. Platelet counts did not change significantly in the LMWH group. None of the patients experienced any adverse effects other than small bruises around the LMWH injection sites. There were three protocol deviations in the control group where LMWH was administrated for patient choice, OHSS and basilic vein thrombosis. Conversely in LMWH group two patients chose not to take the study drug. Demographic characteristics of participants and the features of IVF cycles are presented in Table 1. There were no significant differences between the two group baseline characteristics, neither demographic nor gynecological. Also, in each age we did not find any difference in baseline characteristics. Forty-four women (16.5%) had ≥ 3 previous attempts and twenty-eight patients (10.5%) had one thrombophilic abnormality, FV Leiden being the most common (17 patients). Main outcomes were summarized in Table 2. There were 36 cycles cancelled before ovulation trigger or interrupted after oocyte retrieval (poor response, failed fertilization or cleavage, interruption of transfer, high risk of OHSS): 14 in the heparin-treated group and 22 in the control group (p = 0.152). Heparin-treated group had a similar CPR compared with control (21.5% vs. 26.7%, p = 0.389). The abortion rate was 17.2%: 3 (10.3%) and 8 (22.9%) respectively (p = 0.319). No ectopic pregnancies in either group and 3 (4.9%) fetal or neonatal anomalies, all in the LMWH group, were documented: one therapeutic abortion at the 15th week for cystic hygroma and fetal hydrops in Turner syndrome, one premature twin with a heart defect, one live birth with isolated corpus callosum agenesis. A similar LBR was observed in the heparin-treated group compared with control group (18.5% vs. 20.6% respectively, p = 0.757). The subgroups analysis, ≤ 35, 36–38, 39–40 years, showed the following: comparable CPR (22.5% vs 38.8%, p = 0.124; 21.8% vs 17.3%, p = 0.631; 19.4% vs 23.3%, p = 0.762 respectively) and LBR (16.3% vs 32.7%, p = 0.099; 20.0% vs 13.5%, p = 0.443; 19.4% vs 13.3%, p = 0.731 respectively) in treated vs controls. Sensitivity analyses considering only women with ≥ 3 previous IVF attempts and first enrollment and thrombophilic status gave similar results (data not shown).
were excluded. The control group did not receive any medication other than hormonal treatment. In the study group, platelet count was evaluated on the day of oocyte retrieval and after 7–10 days. 2.6. Outcome measures The primary endpoint was the clinical pregnancy rate (CPR), and the main secondary endpoints were the implantation rate (IR) and live birth rate (LBR). Pregnancy was confirmed by measuring serum betahCG levels 12 days after embryo transfer. Clinical pregnancy was defined as the presence of an embryo with a heartbeat at 6 weeks of gestation; a multiple pregnancy was defined as a gestation with more than one embryo; ongoing pregnancy was defined as pregnancy proceeding beyond the 12th gestational week. LBR was defined as delivery of one or more live infants after 23 gestational weeks. Preterm delivery was defined as delivery before completion of the 37th week of gestation. IR was calculated for each participant as number of gestational sacs divided by number of transferred embryos multiplied by 100 and treated as a continuous variable to account for multiple implantations in a woman. The incidence of major bleeding occurring between intake of the first dose of LMWH and 2 days after the last dose was registered. All the bleeding episodes were collected including minor bleeding. As major bleeding we considered fatal bleedings, bleedings involving a critical organ, or requiring surgery or associated with a decrease in the hemoglobin level of 2 g or more per deciliter or requiring infusion of 2 or more units of blood. Since we studied a population at low risk of bleeding complications (and we had only some small bruises around the LMWH injection site) we did not use any formal definition of clinical relevant non major bleeding event. Other safety outcomes included death, a life-threatening event, persistent or significant disability/incapacity, hospitalization or prolongation of hospitalization, and any other medical condition that may jeopardize the patient or require intervention to prevent the outcomes listed above. 2.7. Statistical analysis and sample size calculation
4. Discussion
Randomization was stratified according to age, considering three age groups: less or equal to 35 years, between 36 and 38 years, and between 39 and 40 years. The CPR was the primary outcome measure. Considering a 5% two-tailed alpha error and a power of 90%, it was calculated that a total of 662 participants should be enrolled in this study. We hypothesized different pregnancy rates in every age class: from 25 to 50% in women ≤35 years (total sample size 170), from 21 to 50% in 36–38 years (total sample size 124), and from 17 to 32% in 39–40 years (total sample size 368). Efficacy analysis was done according to the intention-to-treat principle. Differences between the study groups were assessed using the χ2 test with or without Yates's correction for categorical variables, and independent t-test samples for continuous variables. Sensitivity analyses were planned considering only women with ≥ 3 previous IVF attempts and first enrolment and subgroup analyses according to different age groups (≤ 35, 36–38, and 39–40 years) and to thrombophilic status. All the analyses were performed with Stata 13 (StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP).
In our RCT — the largest so far published — on infertile women undergoing a fresh IVF cycle, the use of parnaparin administered for the whole cycle at a specific dosage according to the patient's body weight did not appear to improve either the CPR or the LBR. Several laboratory studies have hypothesized a positive effect of LMWH in conception and early pregnancy events in infertile women and decrease the risk of thrombosis [6]. Results of clinical studies are inconsistent [16,18,29], and, to date, only one small RCT on patients with a history of three or more previous IVF failures and who had at least one thrombophilic defect demonstrated a significant increase in live births in the heparintreated group compared with the placebo group [16]. On the other hand, use of LMWH started at the time of oocyte retrieval was not associated with a significant increase in the LBR or CPR in two other studies that included a total of about 300 infertile patients [18,29]. In the first of these studies, Urman et al. randomized 150 women with at least 2 failed IVF cycles to an intermediate dose of LMWH up to the 12th gestational week in pregnant participants or no treatment in addition to routine luteal phase support on the day after oocyte retrieval. Noci et al. evaluated whether a prophylactic dose of dalteparin (from the day after the oocyte retrieval and up to the 9th week of pregnancy if positive HCG) could affect IVF outcome in 172 non thrombophilic women < 40 years old. Conversely to literature, in our study we used less strict exclusion criteria, and we included women independently of the number of previous IVF attempts and without only severe thrombophilia. Furthermore we have begun from the hypothesis that the heparan sulphates could play a key role in conception end early pregnancy events and, above all influencing the endometrial receptivity, interacting with many of the known mechanisms that underlie
3. Results Between November 2011 and December 2015, 271 cycles were analyzed in 247 women who agreed to participate in the study: 138 cycles were randomized to LMWH whereas the other 133 constituted the control group. Five cycles were excluded from the primary analysis due to protocol violations (frozen embryos, testicular sperm, intrauterine insemination) leaving 266 cycles for evaluation (135 randomized to LMWH)(Fig. 1). After 4 years, the trial was stopped due to slower enrollment than expected. Twenty-two women underwent more 3
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Fig. 1. CONSORT diagram showing all cycles randomized cycles, excluded and included in the primary analysis allocated in the two arms, and analyzed.
would not achieve a statistical significance. Of note, there was a nonsignificant lower risk of premature interruption of the procedure in the heparin arm, suggesting a potential beneficial effect of LMWH in the earliest phases of IVF, above all in the women 39- to 40-years old. However, this finding was based on a relatively small number of events and should be interpreted with extreme caution. Our study has several potential limitations. First, lack of statistical significance might be due to low statistical power associated with the small global sample size, due to the premature conclusion of the trial. However, considering the results, it is unlikely that, if a beneficial effect of LMWH exists, it could be of clinical relevance in this setting. Furthermore, although we considered only objective endpoints, our study is not double blind. Thus, we could not exclude a different approach in the two groups. Namely, some patients randomized to LMWH could have not taken the treatment during the course study, and patients randomized to the control group may have taken other antithrombotic treatments. Lastly, this study has been conducted in one center only, potentially limiting the external validity of our findings. In conclusion, in our study the administration of a prophylactic dose of LMWH on infertile women undergoing an IVF cycle does not appear to increase the clinical pregnancy or live birth rate. However, LMWH confirmed its safety in this setting, and other larger properly addressed RCTs are still warranted to confirm our findings.
successful apposition, adhesion and penetration of the developing embryo. Biological former studies have clearly demonstrated that these phases fully depending by the hormonal stimulation that has not ruled out with frozen embryos, that we excluded to avoid a fundamental bias. In this way we think that our results are more generalizable to women in the real life. However, the results of sensitivity analysis, which consider only patients with at least three previous IVF attempts, also failed to demonstrate an efficacy of LMWH in this setting. Contrary to the results of the study by Qublan et al. [16], we did not find any effect of LMWH in women with thrombophilia. These conflicting results may be due to the relatively small sample of our thrombophilic patients. However, although some case-control studies have suggested a potential detrimental role of inherited and acquired thrombophilia in this setting, this evidence is inconclusive and not supported by high quality prospective studies [16,30,31]. Our RCT failed to demonstrate a superiority of LMWH in improving CPR, our primary endpoint, in each age group of stratification. We enrolled fewer patients than we had planned. However, on the basis of observed results, we estimated that even with the whole planned sample it would have been unlikely to demonstrate a beneficial effect for LMWH. Specifically in the ≤ 35 years, it's likely we would have had a result significantly against LMWH; in the 36–38 years, where the target sample size was almost reached, and in the 39–40 years the CPR 4
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Table 1 Baseline characteristics.
Enrolled Cycles Age ≤ 35 y 36–38 y 39–40 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 Failed cycles Etiology of infertility Unexplained Male Ovulatory Poor ovarian reserve Tubal factor Endometriosis Mixed female and male factor Multiple female factor FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age ≤ 35 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age 36–38 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age 39–40 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH
Table. 2 Overall outcomes. n
LMWH
Control
266
135
131
P value N retrieval oocytes Cumulative embryo score N Transferred embryos Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age ≤ 35 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age 36–38 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age 39–40 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n
0.987 98 (36.8) 107 (40.2) 61 (22.9) 48 (18.1) 59 (22.2) 89 (33.5) 96 (36.1) 44 (16.5)
49 55 31 28 32 47 43 20
(36.3) (40.7) (23.0) (20.7) (23.7) (34.8) (31.9) (14.8)
49 52 30 20 27 42 53 24
(37.4) (39.7) (22.9) (15.3) (20.6) (32.1) (40.5) (18.3)
23 (8.7) 55 (20.7) 6 (2.3) 46 (17.3) 28 (10.5) 9 (3.4) 77 (29.0)
10 (7.4) 24 (17.8) 2 (1.5) 29 (21.5) 13 (9.6) 6 (4.4) 38 (28.2)
13 (9.9) 31 (23.7) 4 (3.1) 17 (13.0) 15 (11.5) 3 (2.3) 39 (29.8)
22 (8.3) 28 (10.5)
13 (9.6) 13 (9.6)
9 (6.9) 15 (11.5)
2.20 ± 2.78 7.38 ± 3.06 98 11 (11.2) 23 (23.5) 31 (31.6) 39 (39.8) 13 (13.3) 7 (7.1)
1.88 ± 2.18 7.57 ± 3.50 49 6 (12.2) 12 (24.5) 14 (28.6) 16 (32.7) 7 (14.3) 4 (8.2)
2.51 ± 3.26 7.17 ± 2.53 49 5 (10.2) 11 (22.5) 17 (34.7) 23 (46.9) 6 (12.2) 3 (6.1)
2.47 ± 2.63 7.44 ± 3.89 107 26 (24.3) 23 (21.5) 33 (30.8) 38 (35.5) 18 (16.8) 15 (14.0)
2.43 ± 2.50 7.73 ± 4.68 55 17 (30.9) 13 (23.6) 18 (32.7) 21 (38.2) 5 (9.1) 8 (14.6)
2.50 ± 2.78 7.15 ± 2.92 52 9 (17.3) 10 (19.2) 15 (28.9) 17 (32.7) 13 (25.0) 7 (13.5)
1.95 ± 2.65 7.63 ± 2.41 61 11 (18.0) 13 (21.3) 25 (41.0) 19 (31.2) 13 (21.3) 6 (9.8)
1.46 ± 1.68 7.74 ± 2.52 31 5 (16.1) 7 (22.6) 15 (48.4) 6 (19.4) 8 (25.8) 1 (3.2)
2.46 ± 3.33 7.52 ± 2.30 30 6 (20.0) 6 (20.0) 10 (33.3) 13 (43.3) 5 (16.7) 5 (16.7)
0.749 1.000 0.300 0.056 0.534 0.104
2.21 ± 3.21 6.82 ± 2.49
1.82 ± 2.34 7.03 ± 2.79
2.61 ± 3.91 6.60 ± 2.13
0.868 0.639
0.267 0.559 0.697 0.161 0.510 0.447
0.692
0.076 0.458 1.000 1.000 0.664 0.215 1.000 1.000
0.782 0.654 0.118 0.642 0.681 0.686 0.038 1.000
Total
LMWH
Control
P value
9.05 ± 5.14 4.22 ± 1.76
8.55 ± 4.12 4.25 ± 1.76
9.55 ± 5.96 4.19 ± 1.76
0.559 0.748
1.94 ± 0.77 64 (24.1) 52 (19.6) 16.2 ± 31.2 11 (17.2) 1 98 10.1 ± 5.3 3.8 ± 1.3
2.02 ± 0.69 29 (21.5) 25 (18.5) 13.7 ± 28.2 3 (10.3) 1 49 9.4 ± 4.3 3.7 ± 1.2
1.85 ± 0.84 35 (26.7) 27 (20.6) 18.8 ± 33.9 8 (22.9) 0 49 10.7 ± 60 3.9 ± 1.4
0.165 0.389 0.757 0.262 0.319 NA 0.337 0.504
30 (30.6) 24 (24.5) 22.4 ± 36.7 5 (16.7) 1
11 (22.5) 8 (16.3) 15.3 ± 31.0 2 (18.2) 1
19 (38.8) 16 (32.7) 29.6 ± 40.7 3 (15.8) 0
0.124 0.099 0.062 1.000 NA
107 8.4 ± 5.3 4.1 ± 1.8
55 8.1 ± 4.3 4.0 ± 1.8
52 8.8 ± 6.1 4.1 ± 1.8
0.849 0.882
21 (19.6) 18 (16.8) 12.3 ± 26.9 3 (14.3%) 0
12 (21.8) 11 (20.0) 13.3 ± 26.9 1 (8.3%) 0
9 (17.3) 7 (13.5) 11.2 ± 27.2 2 (22.2%) 0
0.631 0.443 0.562 0.533 NA
61 8.5 ± 4.4 5.2 ± 2.0
31 8.1 ± 3.3 5.4 ± 1.9
30 8.9 ± 5.3 4.9 ± 2.1
0.667 0.315
13 (21.3) 10 (16.4) 13.1 ± 27.2 3 (23.1) 0
6 (19.4) 6 (19.4) 11.8 ± 26.6 0 0
7 (23.3) 4 (13.3) 14.4 ± 28.3 3 (42.9) 0
0.762 0.731 0.709 0.192 NA
NA: not applicable.
Declaration of interests The authors state that they have no conflict of interest.
0.028 0.729
Funding This work was partially supported by Alfa Wassermann S.p.A. Acknowledgments The authors thank all of the patients who participated in this study; Alfa Wassermann S.p.A. for his economic support and for the drug supply and Fleming Research S.R.L. for the analysis of genetic thrombophilia; Alba Tocchella who served as nurse in the blood collection.
Contributors
Appendix A. Supplementary data
C. Lodigiani, E. Banfi, P. E. Levi Setti designed the study; P. Ferrazzi, L. Librè, L. Cafaro, V. Pacetti, E. Zannoni, A.M. Baggiani recruited and supervised recruitment of patients; E. Banfi randomized patients and supervised data collection; I. Quaglia undertook all assays; E. Morenghi, F. Dentali analyzed the data; C. Lodigiani, F. Dentali, P.E. Levi Setti wrote the first draft and all authors contributed the final manuscript. C. Lodigiani takes responsibility of the study.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2017.08.006. References [1] P.C. Steptoe, R.G. Edwards, Birth after the reimplantation of a human embryo, Lancet 2 (1978) 366. [2] M. Donaghy, B.A. Lessey, Uterine receptivity: alternations associated with benign gynecologic disease, Semin. Reprod. Med. 25 (2007) 461–475. [3] A.N. Anderson, V. Goossens, A.P. Ferraretti, et al., European IVF-monitoring (EIM) Consortium, European Society of Human Reproduction and Embryology (ESHRE),
5
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Assisted reproductive technology in Europe, 2004: results generated from European registers by ESHRE, Hum. Reprod. 23 (2008) 756–771. R.T. Scott Jr., J.M. Franasiak, E.J. Forman, Comprehensive chromosome screening with synchronous blastocyst transfer: time for a paradigm shift, Fertil. Steril. 102 (2014) 660–661. E. Grandone, M. Villani, F. Dentali, et al., Low-molecular-weight heparin in pregnancies after ART - a retrospective study, Thromb. Res. 134 (2014) 336–339. S.M. Nelson, I.A. Greer, The potential role of heparin in assisted conception, Hum. Reprod. Update 14 (2008) 623–645. N. Di Simone, F. Di Nicuolo, M. Sanguinetti, et al., Low-molecular weight heparin induces in vitro trophoblast invasiveness: role of matrix metalloproteinases and tissue inhibitors, Placenta 28 (2007) 298–304. O. Erden, A. Imir, T. Guvenal, et al., Investigation of the effects of heparin and low molecular weight heparin on E-cadherin and laminin expression in rat pregnancy by immunohistochemistry, Hum. Reprod. 21 (2006) 3014–3018. IeM Shih, M.Y. Hsu, R.J. Oldt 3rdet al., The role of E-cadherin in the motility and invasion of implantation site intermediate trophoblast, Placenta 23 (2002) 706–715. S.K. Das, X.N. Wang, B.C. Paria, et al., Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation, Development 120 (1994) 1071–1083. R.E. Leach, B. Kilburn, J. Wang, et al., Heparin-binding EGF-like growth factor regulates human extravillous cytotrophoblast development during conversion to the invasive phenotype, Dev. Biol. 266 (2004) 223–237. H. Lacey, T. Haigh, M. Westwood, et al., Mesenchymally-derived insulin-like growth factor 1 provides a paracrine stimulus for trophoblast migration, BMC Dev. Biol. 2 (2002) 5. W.H. Kutteh, D.L. Yetman, S.J. Chantilis, et al., Effect of antiphospholipid antibodies in women undergoing in-vitro fertilization: role of heparin and aspirin, Hum. Reprod. 12 (1997) 1171–1175. G. Sher, C. Zouves, M. Feinman, et al., A rational basis for the use of combined heparin/aspirin and IVIG immunotherapy in the treatment of recurrent IVF failure associated with antiphospholipid antibodies, Am. J. Reprod. Immunol. 39 (1998) 391–394. C. Stern, L. Chamley, Antiphospholipid antibodies and coagulation defects in women with implantation failure after IVF and recurrent miscarriage, Reprod. BioMed. Online 13 (2006) 29–37. H. Qublan, Z. Amarin, M. Dabbas, et al., Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial, Hum. Fertil. 11 (2008) 246–253. B. Berker, S. Taşkin, K. Kahraman, et al., The role of low-molecular-weight heparin
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Full Length Article
The effect of parnaparin sodium on in vitro fertilization outcome: A prospective randomized controlled trial Corrado Lodigiania,⁎, Francesco Dentalib, Elena Banfia, Paola Ferrazzia, Luca Librèa, Ilaria Quagliaa, Luca Cafaroc, Emanuela Morenghid, Veronica Pacettia, Elena Zannonic, Anna Maria Baggianic, Paolo Emanuele Levi-Settic a
Humanitas Research Hospital, Cardiovascular Department, Thrombosis and Haemorragic Diseases Center, Rozzano, Milan, Italy Department of Clinical and Experimental Medicine, Insubria University, Varese, Italy c Humanitas Research Hospital, Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Fertility Center, Rozzano, Milan, Italy. d Humanitas Research Hospital, Biostatistics Unit, Rozzano, Milan, Italy b
A R T I C L E I N F O
A B S T R A C T
Keywords: In vitro fertilization Live-birth pregnancy rate Low-molecular weight heparin Reproductive sterility Thrombophilia
Introduction: In-vitro and in-vivo models suggest the influence of low-molecular weight heparin on conception in infertile women undergoing in vitro fertilization procedures (IVF). In this randomized controlled trial we assessed whether a low-molecular weight heparin (parnaparin) could affect IVF outcomes. Materials and methods: 271 cycles were analyzed in 247 women having a first or subsequent IVF cycle at Fertility Center of Humanitas Research Hospital. Patients, without severe thrombophilia and hormonal or active untreated autoimmune disorders, were randomly allocated (1:1) to receive for the whole cycle parnaparin, or routine hormonal therapy only. The primary endpoint was the clinical pregnancy rate and the secondary endpoints included implantation rate and live birth rate. Results: The clinical pregnancy and the live birth rate were similar in treated and controls (21.5% vs. 26.7%, p = 0.389; 18.5% vs. 20.6%, p = 0.757). The abortion rate was 10.3% vs 22.9%, p = 0.319, respectively. The subgroups analysis, ≤ 35, 36–38, 39–40 years, showed the following: comparable clinical pregnancy rate (22.5% vs 38.8%, p = 0.124; 21.8% vs 17.3%, p = 0.631; 19.4% vs 23.3%, p = 0.762 respectively) and live birth rate (16.3% vs 32.7%, p = 0.099; 20.0% vs 13.5%, p = 0.443; 19.4% vs 13.3%, p = 0.731 respectively) in treated vs controls. Sensitivity analyses on women with ≥ 3 previous attempts and first enrolment only, and subgroup analyses according to trial conclusion conditioning a small sample size with low statistical power. Conclusions: Our study excludes positive effect of parnaparin, once a day for the whole cycle, on clinical pregnancy rate in infertile women undergoing in vitro fertilization techniques.
1. Introduction Since the introduction into clinical practice of in vitro fertilization procedures (IVF) [1], improvements have been made to ovarian stimulation protocols, preparation of follicles and gamete culture medium. However, the implantation rates of the pre-embryo are still low [2], which is the actual limiting factor for the success of IVF in terms of live birth rate [3]. Since embryo implantation depends on several factors, the causes of its failure, even if the embryo is transferred at blastocyst stage after full chromosomal analysis [4], remain unknown. Unfortunately, this may occasionally be a recurring phenomenon leading to despair in couples and frustration in their caregivers. Recently, some experts have advocated the use of antithrombotic drugs (heparins and aspirin) in order to improve the implantation rate [5]. ⁎
Heparin can have a positive effect in conception and early pregnancy events by altering the hemostatic response to ovarian stimulation, modulating trophoblast differentiation and invasion, and decreasing the risk of thrombosis [6]. In-vitro and in-vivo models suggest the influence of low-molecular weight heparin (LMWH) on different aspects of trophoblast adhesion and invasiveness by acting on matrix metalloproteinases and tissue inhibitors [7], cadherin-E [8,9], heparin-binding epidermal growth factor [10,11], and free insulin-like growth factor [6,12]. Clinical studies suggest an influence of heparin on IVF outcome under specific clinical conditions such as women affected by antiphospholipid antibodies (aPL) [13,14,15]. The role of heparin in patients with recurrent IVF failure without an aPL syndrome is more conflicting [16,17]. In general, original studies gave negative results. A comprehensive systematic review and meta-analysis of published literature on
Corresponding author at: Humanitas Research Hospital, Cardiovascular Department, Thrombosis and Haemorragic Diseases Center, via Manzoni 56, 20089 Rozzano, Milan, Italy. E-mail address: [email protected] (C. Lodigiani).
http://dx.doi.org/10.1016/j.thromres.2017.08.006 Received 16 June 2017; Received in revised form 7 August 2017; Accepted 13 August 2017 0049-3848/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Lodigiani, C., Thrombosis Research (2017), http://dx.doi.org/10.1016/j.thromres.2017.08.006
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a contraindication for heparin therapy were excluded. Patients with a family history (even strong) of thrombosis were not excluded from the study. According to the study protocol, patients could be enrolled in the study more than once. Participating women were submitted to a block computer-generated randomization and allocated to receive or not to receive parnaparin for the whole cycle at a daily dose of 4250 anti-Xa IU/0.4 mL and 6400 anti-Xa IU/0.6 mL, if the body weight was respectively under or over 60 kg. The randomization list was prepared by a biostatistician not involved in patient recruitment. Opaque numbered and sealed envelopes containing the allocation were managed by the study data manager, who informed the attending physicians. Data were collected on BMI, smoking habits, infertility causes, previous pregnancies, miscarriages, and IVF procedures, retrieved oocytes, transferred embryos and implantation rate, IVF outcome and parameters used for randomization.
the effect of heparin on the outcome of IVF was conducted by Seshadri et al. in 2012 which included both randomized and observational studies. The randomized studies included only small numbers of women, and had high methodological heterogeneity, and so had significant limitations. These showed no significant difference in implantation rate, clinical pregnancy, clinical miscarriage and live birth rate. Metaanalysis of the observational studies showed a significant increase in the clinical pregnancy rate and live birth rate, however the authors concluded that the potential role of heparin during IVF treatment required further evaluation in adequately powered randomized studies as they noted that the observational studies could have exaggerated the value of heparin in IVF due to selection bias. In a more recent metaanalysis, the use of LMWH significantly increased the clinical pregnancy rate and live birth rate [18,19,20]. Despite the absence of robust clinical evidence, maternal LMWH administration is used in IVF as an intervention that may improve implantation and reduce miscarriage, although clinical practice varies widely between individual clinicians and clinics. Thus, we conducted a randomized controlled trial (RCT) to assess whether LMWH administration in infertile women having a first or subsequent IVF cycle could contribute to improving the clinical outcome, with or without the presence of non-severe thrombophilia.
2.4. Laboratory tests Factor VG1691A and FIIG20210A were assessed with HTRBio-toBit-PCR Platform, Fleming Research. The activities of anticoagulant proteins were measured in human citrated plasma on IL Coagulation System® (Instrumentation Laboratory, Bedford, USA): protein C and antithrombin with an automated chromogenic assay; free protein S with automated latex ligand immunoassay; resistance to activated protein C with COATEST™ APC RESISTENCE™. Lupus anticoagulant was determined with one-stage clotting test using Simplified Dilute Russel Viper Venom (IL). Anti-Cardiolipin IgG/IgM antibodies were quantified in human serum in accordance with manufacturer's instructions (ORGENTEC, Diagnostika GmbH, Germany).
2. Materials and methods 2.1. Study design This randomized, prospective, controlled, stratified, open label and phase III study was carried out and reported according to the CONSORT (Consolidated Standards of Reporting Trials) guidelines for randomized controlled studies [21]. The study was approved by the institutional review board of our Institute and written informed consent was obtained for all women and is registered on ClinicalTrials.gov NCT02991950.
2.5. Interventions During the study period, patients underwent a controlled ovarian hyperstimulation (COH) protocol. The ovarian stimulation protocol was determined according to ovarian reserve parameters, evaluating antiMüllerian hormone (AMH), antral follicle count (AFC), and body mass index (BMI) before the treatment [22,23] and considering previous treatment cycles [24,25]. COH was performed with a GnRH agonist (Enantone die, Takeda, Italy or Triptorelin Depot 3.75 mg IM, Decapeptyl®, Ipsen, Milan, Italy) or GnRH antagonist protocol (Cetrotide®, Merck Serono, Rome, Italy; Orgalutran®, Organon, MSD-Italy) [26]. In the GnRH, agonist protocol induction was started after evidence of pituitary desensitization (absence of ovarian follicles > 10 mm and endometrial thickness < 5.4 mm on transvaginal ultrasound examination). In the GnRH antagonist protocol, women received a low dose oral contraceptive and gonadotrophins were started the first day of withdrawal bleeding [27]. The starting dose of rFSH (Puregon®, MSD-Italy; Gonal-F, Serono, Rome, Italy) or hMG (Meropur®, Ferring, Milan, Italy) was decided according to ovarian reserve parameters and or previous induction cycle for the first four days and thereafter, on the basis of transvaginal follicular parameters (number and diameter) and results of estradiol and progesterone determination, a variable dose of gonadotropin was administered until the day of ovulation trigger with a 250 mcg rhCG (Ovitrelle®, Serono, Rome, Italy Merck Serono) injection. Parnaparin sodium (Fluxum 4250 anti-Xa IU/0.4 mL or 6400 antiXa IU/0.6 mL; Alfa Wassermann S.p.A., Bologna, Italy) was administered for the whole cycle, from the day before the beginning of the stimulation phase of the cycle until the result of the procedure and, in the case of evolutive pregnancy, until delivery or the end of pregnancy. According to the study protocol, LMWH was discontinued in the case of patient refusal, toxicity or other complications precluding further therapy, or in the case of a decision by the physician. During LMWH administration, concomitant therapy with acetylsalicylic acid or steroids was not allowed, so patients on chronic treatment with these drugs
2.2. Aim of the study To assess whether a low molecular weight heparin, parnaparin, administration could contribute to improving the outcome of IVF in terms of clinical pregnancy rate in infertile women undergoing a cycle of IVF, aged ≤40 years and ≥18 years, without severe thrombophilia, and hormonal or active untreated autoimmune disorders. 2.3. Patients and methods The study was undertaken at the Fertility Center and Thrombosis and Haemorragic Diseases Center of Humanitas Research Hospital, Milan, Italy, between November 2011 to December 2015. The last participant was recruited in April 2015, and the follow-up was completed in December 2015. Originally we planned the conduct the study as a multicenter study. Subsequently it was amended as single center study due to the high management of costs. Infertile women satisfying all the inclusion and exclusion criteria were potentially eligible for the study. Women between 18 and 40 years of age who underwent a fresh IVF cycle with routine ovulation induction protocol were candidates for the study. Cycles with frozen ejaculated or testicular sperm and cycles with frozen embryos were excluded from the study, in order to include a quite homogeneous population with exclusion of all possible bias (e.g. severe male factor leading to the use of frozen or testicular sperm). Women with chronic disease (liver, renal, thyroid) or hormonal disorder not compensated with a specific therapy, with a immunological disease (e.g., autoimmune thyroiditis, connectivitis, rheumatoid arthritis, systemic lupus erythematosus), with abnormal platelet count (< 100,000/mm3), with antiphospholipid autoantibodies or other severe thrombophilia (antithrombin, protein S, protein C deficiency or homozygous FV Leiden or FIIG20210A, or double heterozygous FV Leiden and FIIG20210A), and patients with previous thrombosis or with 2
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than one IVF during the study period and were included more than once in the study. No woman was lost at follow-up. Platelet counts did not change significantly in the LMWH group. None of the patients experienced any adverse effects other than small bruises around the LMWH injection sites. There were three protocol deviations in the control group where LMWH was administrated for patient choice, OHSS and basilic vein thrombosis. Conversely in LMWH group two patients chose not to take the study drug. Demographic characteristics of participants and the features of IVF cycles are presented in Table 1. There were no significant differences between the two group baseline characteristics, neither demographic nor gynecological. Also, in each age we did not find any difference in baseline characteristics. Forty-four women (16.5%) had ≥ 3 previous attempts and twenty-eight patients (10.5%) had one thrombophilic abnormality, FV Leiden being the most common (17 patients). Main outcomes were summarized in Table 2. There were 36 cycles cancelled before ovulation trigger or interrupted after oocyte retrieval (poor response, failed fertilization or cleavage, interruption of transfer, high risk of OHSS): 14 in the heparin-treated group and 22 in the control group (p = 0.152). Heparin-treated group had a similar CPR compared with control (21.5% vs. 26.7%, p = 0.389). The abortion rate was 17.2%: 3 (10.3%) and 8 (22.9%) respectively (p = 0.319). No ectopic pregnancies in either group and 3 (4.9%) fetal or neonatal anomalies, all in the LMWH group, were documented: one therapeutic abortion at the 15th week for cystic hygroma and fetal hydrops in Turner syndrome, one premature twin with a heart defect, one live birth with isolated corpus callosum agenesis. A similar LBR was observed in the heparin-treated group compared with control group (18.5% vs. 20.6% respectively, p = 0.757). The subgroups analysis, ≤ 35, 36–38, 39–40 years, showed the following: comparable CPR (22.5% vs 38.8%, p = 0.124; 21.8% vs 17.3%, p = 0.631; 19.4% vs 23.3%, p = 0.762 respectively) and LBR (16.3% vs 32.7%, p = 0.099; 20.0% vs 13.5%, p = 0.443; 19.4% vs 13.3%, p = 0.731 respectively) in treated vs controls. Sensitivity analyses considering only women with ≥ 3 previous IVF attempts and first enrollment and thrombophilic status gave similar results (data not shown).
were excluded. The control group did not receive any medication other than hormonal treatment. In the study group, platelet count was evaluated on the day of oocyte retrieval and after 7–10 days. 2.6. Outcome measures The primary endpoint was the clinical pregnancy rate (CPR), and the main secondary endpoints were the implantation rate (IR) and live birth rate (LBR). Pregnancy was confirmed by measuring serum betahCG levels 12 days after embryo transfer. Clinical pregnancy was defined as the presence of an embryo with a heartbeat at 6 weeks of gestation; a multiple pregnancy was defined as a gestation with more than one embryo; ongoing pregnancy was defined as pregnancy proceeding beyond the 12th gestational week. LBR was defined as delivery of one or more live infants after 23 gestational weeks. Preterm delivery was defined as delivery before completion of the 37th week of gestation. IR was calculated for each participant as number of gestational sacs divided by number of transferred embryos multiplied by 100 and treated as a continuous variable to account for multiple implantations in a woman. The incidence of major bleeding occurring between intake of the first dose of LMWH and 2 days after the last dose was registered. All the bleeding episodes were collected including minor bleeding. As major bleeding we considered fatal bleedings, bleedings involving a critical organ, or requiring surgery or associated with a decrease in the hemoglobin level of 2 g or more per deciliter or requiring infusion of 2 or more units of blood. Since we studied a population at low risk of bleeding complications (and we had only some small bruises around the LMWH injection site) we did not use any formal definition of clinical relevant non major bleeding event. Other safety outcomes included death, a life-threatening event, persistent or significant disability/incapacity, hospitalization or prolongation of hospitalization, and any other medical condition that may jeopardize the patient or require intervention to prevent the outcomes listed above. 2.7. Statistical analysis and sample size calculation
4. Discussion
Randomization was stratified according to age, considering three age groups: less or equal to 35 years, between 36 and 38 years, and between 39 and 40 years. The CPR was the primary outcome measure. Considering a 5% two-tailed alpha error and a power of 90%, it was calculated that a total of 662 participants should be enrolled in this study. We hypothesized different pregnancy rates in every age class: from 25 to 50% in women ≤35 years (total sample size 170), from 21 to 50% in 36–38 years (total sample size 124), and from 17 to 32% in 39–40 years (total sample size 368). Efficacy analysis was done according to the intention-to-treat principle. Differences between the study groups were assessed using the χ2 test with or without Yates's correction for categorical variables, and independent t-test samples for continuous variables. Sensitivity analyses were planned considering only women with ≥ 3 previous IVF attempts and first enrolment and subgroup analyses according to different age groups (≤ 35, 36–38, and 39–40 years) and to thrombophilic status. All the analyses were performed with Stata 13 (StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP).
In our RCT — the largest so far published — on infertile women undergoing a fresh IVF cycle, the use of parnaparin administered for the whole cycle at a specific dosage according to the patient's body weight did not appear to improve either the CPR or the LBR. Several laboratory studies have hypothesized a positive effect of LMWH in conception and early pregnancy events in infertile women and decrease the risk of thrombosis [6]. Results of clinical studies are inconsistent [16,18,29], and, to date, only one small RCT on patients with a history of three or more previous IVF failures and who had at least one thrombophilic defect demonstrated a significant increase in live births in the heparintreated group compared with the placebo group [16]. On the other hand, use of LMWH started at the time of oocyte retrieval was not associated with a significant increase in the LBR or CPR in two other studies that included a total of about 300 infertile patients [18,29]. In the first of these studies, Urman et al. randomized 150 women with at least 2 failed IVF cycles to an intermediate dose of LMWH up to the 12th gestational week in pregnant participants or no treatment in addition to routine luteal phase support on the day after oocyte retrieval. Noci et al. evaluated whether a prophylactic dose of dalteparin (from the day after the oocyte retrieval and up to the 9th week of pregnancy if positive HCG) could affect IVF outcome in 172 non thrombophilic women < 40 years old. Conversely to literature, in our study we used less strict exclusion criteria, and we included women independently of the number of previous IVF attempts and without only severe thrombophilia. Furthermore we have begun from the hypothesis that the heparan sulphates could play a key role in conception end early pregnancy events and, above all influencing the endometrial receptivity, interacting with many of the known mechanisms that underlie
3. Results Between November 2011 and December 2015, 271 cycles were analyzed in 247 women who agreed to participate in the study: 138 cycles were randomized to LMWH whereas the other 133 constituted the control group. Five cycles were excluded from the primary analysis due to protocol violations (frozen embryos, testicular sperm, intrauterine insemination) leaving 266 cycles for evaluation (135 randomized to LMWH)(Fig. 1). After 4 years, the trial was stopped due to slower enrollment than expected. Twenty-two women underwent more 3
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Fig. 1. CONSORT diagram showing all cycles randomized cycles, excluded and included in the primary analysis allocated in the two arms, and analyzed.
would not achieve a statistical significance. Of note, there was a nonsignificant lower risk of premature interruption of the procedure in the heparin arm, suggesting a potential beneficial effect of LMWH in the earliest phases of IVF, above all in the women 39- to 40-years old. However, this finding was based on a relatively small number of events and should be interpreted with extreme caution. Our study has several potential limitations. First, lack of statistical significance might be due to low statistical power associated with the small global sample size, due to the premature conclusion of the trial. However, considering the results, it is unlikely that, if a beneficial effect of LMWH exists, it could be of clinical relevance in this setting. Furthermore, although we considered only objective endpoints, our study is not double blind. Thus, we could not exclude a different approach in the two groups. Namely, some patients randomized to LMWH could have not taken the treatment during the course study, and patients randomized to the control group may have taken other antithrombotic treatments. Lastly, this study has been conducted in one center only, potentially limiting the external validity of our findings. In conclusion, in our study the administration of a prophylactic dose of LMWH on infertile women undergoing an IVF cycle does not appear to increase the clinical pregnancy or live birth rate. However, LMWH confirmed its safety in this setting, and other larger properly addressed RCTs are still warranted to confirm our findings.
successful apposition, adhesion and penetration of the developing embryo. Biological former studies have clearly demonstrated that these phases fully depending by the hormonal stimulation that has not ruled out with frozen embryos, that we excluded to avoid a fundamental bias. In this way we think that our results are more generalizable to women in the real life. However, the results of sensitivity analysis, which consider only patients with at least three previous IVF attempts, also failed to demonstrate an efficacy of LMWH in this setting. Contrary to the results of the study by Qublan et al. [16], we did not find any effect of LMWH in women with thrombophilia. These conflicting results may be due to the relatively small sample of our thrombophilic patients. However, although some case-control studies have suggested a potential detrimental role of inherited and acquired thrombophilia in this setting, this evidence is inconclusive and not supported by high quality prospective studies [16,30,31]. Our RCT failed to demonstrate a superiority of LMWH in improving CPR, our primary endpoint, in each age group of stratification. We enrolled fewer patients than we had planned. However, on the basis of observed results, we estimated that even with the whole planned sample it would have been unlikely to demonstrate a beneficial effect for LMWH. Specifically in the ≤ 35 years, it's likely we would have had a result significantly against LMWH; in the 36–38 years, where the target sample size was almost reached, and in the 39–40 years the CPR 4
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Table 1 Baseline characteristics.
Enrolled Cycles Age ≤ 35 y 36–38 y 39–40 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 Failed cycles Etiology of infertility Unexplained Male Ovulatory Poor ovarian reserve Tubal factor Endometriosis Mixed female and male factor Multiple female factor FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age ≤ 35 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age 36–38 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH Age 39–40 y BMI > 25 kg/m2 Active Smoke Previous pregnancy First attempt ≥ 3 (Failed) cycles FVG1691A or FIIG20210A Heterozygous Basal AMH Basal FSH
Table. 2 Overall outcomes. n
LMWH
Control
266
135
131
P value N retrieval oocytes Cumulative embryo score N Transferred embryos Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age ≤ 35 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age 36–38 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n Age 39–40 y N retrieval oocytes Cumulative embryo score Clinical Pregnancy, n Live birth, n Implantation Rate, % Abortion, n Therapeutic abortion, n
0.987 98 (36.8) 107 (40.2) 61 (22.9) 48 (18.1) 59 (22.2) 89 (33.5) 96 (36.1) 44 (16.5)
49 55 31 28 32 47 43 20
(36.3) (40.7) (23.0) (20.7) (23.7) (34.8) (31.9) (14.8)
49 52 30 20 27 42 53 24
(37.4) (39.7) (22.9) (15.3) (20.6) (32.1) (40.5) (18.3)
23 (8.7) 55 (20.7) 6 (2.3) 46 (17.3) 28 (10.5) 9 (3.4) 77 (29.0)
10 (7.4) 24 (17.8) 2 (1.5) 29 (21.5) 13 (9.6) 6 (4.4) 38 (28.2)
13 (9.9) 31 (23.7) 4 (3.1) 17 (13.0) 15 (11.5) 3 (2.3) 39 (29.8)
22 (8.3) 28 (10.5)
13 (9.6) 13 (9.6)
9 (6.9) 15 (11.5)
2.20 ± 2.78 7.38 ± 3.06 98 11 (11.2) 23 (23.5) 31 (31.6) 39 (39.8) 13 (13.3) 7 (7.1)
1.88 ± 2.18 7.57 ± 3.50 49 6 (12.2) 12 (24.5) 14 (28.6) 16 (32.7) 7 (14.3) 4 (8.2)
2.51 ± 3.26 7.17 ± 2.53 49 5 (10.2) 11 (22.5) 17 (34.7) 23 (46.9) 6 (12.2) 3 (6.1)
2.47 ± 2.63 7.44 ± 3.89 107 26 (24.3) 23 (21.5) 33 (30.8) 38 (35.5) 18 (16.8) 15 (14.0)
2.43 ± 2.50 7.73 ± 4.68 55 17 (30.9) 13 (23.6) 18 (32.7) 21 (38.2) 5 (9.1) 8 (14.6)
2.50 ± 2.78 7.15 ± 2.92 52 9 (17.3) 10 (19.2) 15 (28.9) 17 (32.7) 13 (25.0) 7 (13.5)
1.95 ± 2.65 7.63 ± 2.41 61 11 (18.0) 13 (21.3) 25 (41.0) 19 (31.2) 13 (21.3) 6 (9.8)
1.46 ± 1.68 7.74 ± 2.52 31 5 (16.1) 7 (22.6) 15 (48.4) 6 (19.4) 8 (25.8) 1 (3.2)
2.46 ± 3.33 7.52 ± 2.30 30 6 (20.0) 6 (20.0) 10 (33.3) 13 (43.3) 5 (16.7) 5 (16.7)
0.749 1.000 0.300 0.056 0.534 0.104
2.21 ± 3.21 6.82 ± 2.49
1.82 ± 2.34 7.03 ± 2.79
2.61 ± 3.91 6.60 ± 2.13
0.868 0.639
0.267 0.559 0.697 0.161 0.510 0.447
0.692
0.076 0.458 1.000 1.000 0.664 0.215 1.000 1.000
0.782 0.654 0.118 0.642 0.681 0.686 0.038 1.000
Total
LMWH
Control
P value
9.05 ± 5.14 4.22 ± 1.76
8.55 ± 4.12 4.25 ± 1.76
9.55 ± 5.96 4.19 ± 1.76
0.559 0.748
1.94 ± 0.77 64 (24.1) 52 (19.6) 16.2 ± 31.2 11 (17.2) 1 98 10.1 ± 5.3 3.8 ± 1.3
2.02 ± 0.69 29 (21.5) 25 (18.5) 13.7 ± 28.2 3 (10.3) 1 49 9.4 ± 4.3 3.7 ± 1.2
1.85 ± 0.84 35 (26.7) 27 (20.6) 18.8 ± 33.9 8 (22.9) 0 49 10.7 ± 60 3.9 ± 1.4
0.165 0.389 0.757 0.262 0.319 NA 0.337 0.504
30 (30.6) 24 (24.5) 22.4 ± 36.7 5 (16.7) 1
11 (22.5) 8 (16.3) 15.3 ± 31.0 2 (18.2) 1
19 (38.8) 16 (32.7) 29.6 ± 40.7 3 (15.8) 0
0.124 0.099 0.062 1.000 NA
107 8.4 ± 5.3 4.1 ± 1.8
55 8.1 ± 4.3 4.0 ± 1.8
52 8.8 ± 6.1 4.1 ± 1.8
0.849 0.882
21 (19.6) 18 (16.8) 12.3 ± 26.9 3 (14.3%) 0
12 (21.8) 11 (20.0) 13.3 ± 26.9 1 (8.3%) 0
9 (17.3) 7 (13.5) 11.2 ± 27.2 2 (22.2%) 0
0.631 0.443 0.562 0.533 NA
61 8.5 ± 4.4 5.2 ± 2.0
31 8.1 ± 3.3 5.4 ± 1.9
30 8.9 ± 5.3 4.9 ± 2.1
0.667 0.315
13 (21.3) 10 (16.4) 13.1 ± 27.2 3 (23.1) 0
6 (19.4) 6 (19.4) 11.8 ± 26.6 0 0
7 (23.3) 4 (13.3) 14.4 ± 28.3 3 (42.9) 0
0.762 0.731 0.709 0.192 NA
NA: not applicable.
Declaration of interests The authors state that they have no conflict of interest.
0.028 0.729
Funding This work was partially supported by Alfa Wassermann S.p.A. Acknowledgments The authors thank all of the patients who participated in this study; Alfa Wassermann S.p.A. for his economic support and for the drug supply and Fleming Research S.R.L. for the analysis of genetic thrombophilia; Alba Tocchella who served as nurse in the blood collection.
Contributors
Appendix A. Supplementary data
C. Lodigiani, E. Banfi, P. E. Levi Setti designed the study; P. Ferrazzi, L. Librè, L. Cafaro, V. Pacetti, E. Zannoni, A.M. Baggiani recruited and supervised recruitment of patients; E. Banfi randomized patients and supervised data collection; I. Quaglia undertook all assays; E. Morenghi, F. Dentali analyzed the data; C. Lodigiani, F. Dentali, P.E. Levi Setti wrote the first draft and all authors contributed the final manuscript. C. Lodigiani takes responsibility of the study.
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