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Corresponding author: Sofie Bliddal, Department of Medical Endocrinology and Metabolism, section 2132, Copenhagen University Hospital (Rigshospitalet), Blegdamsvej 9, 2100 Copenhagen, Denmark, Phone: +4520762753
Department of Medical Endocrinology and Metabolism, Copenhagen University Hospital (Rigshospitalet), 2100 Copenhagen, DenmarkDepartment of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
Recurrent Pregnancy Loss Unit, Copenhagen University Hospital (Rigshospitalet and Hvidovre Hospital), 2100 Copenhagen, DenmarkDepartment of Gynecology and Obstetrics, Aalborg University Hospital, 9000 Aalborg, Denmark
Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital (Rigshospitalet), 2100 Copenhagen, Denmark
Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, DenmarkRecurrent Pregnancy Loss Unit, Copenhagen University Hospital (Rigshospitalet and Hvidovre Hospital), 2100 Copenhagen, Denmark
Is anti-Müllerian Hormone (AMH) associated with live birth rate in women with unexplained recurrent pregnancy loss (RPL)?
Design
Cohort study of women with unexplained RPL attending the RPL Unit, Copenhagen University Hospital, Denmark, 2015-2021. AMH concentration was assessed upon referral, and live birth rate in the next pregnancy. RPL was defined as ≥3 consecutive pregnancy losses. Regression analyses were adjusted for age, number of previous losses, body mass index, smoking, assisted reproductive technology (ART), and RPL treatments.
Results
A total of 629 women were included, of whom 507 (80.6%) became pregnant after referral. Pregnancy rates were similar for women with low and high AMH compared to women with medium AMH (81.9%, 80.3%, and 79.7%, respectively (low AMH adjusted odds ratio (aOR) 1.44, 95% Confidence Interval (CI):0.84-2.47, p=0.18, and high AMH: aOR 0.89, 95%CI:0.59-1.64, p=0.95). AMH concentrations were not associated with live birth. Live birth rate was 59.5% in women with low, 66.1% with medium, and 65.1% with high AMH (aOR low AMH 0.69, 95%CI:0.42-1.13, p=0.14, aOR high AMH 0.96, 95%CI:0.59-1.56, p=0.96). Live birth was lower in ART pregnancies (aOR 0.56 95%CI:0.33-0.96, p=0.03), and with higher numbers of previous losses (aOR 0.80 95%CI:0.68-0.95, p=0.01).
Conclusion
In women with unexplained RPL, AMH was not associated with the chance of live birth in the next pregnancy. Screening for AMH in all women with RPL is not supported by current evidence. The chance of live birth among women with unexplained RPL achieving pregnancy by ART was low and needs to be confirmed and explored in future studies.
Anti-Müllerian Hormones (AMH) are well-investigated in infertile women, but less so in women with recurrent pregnancy loss (RPL). Lower AMH levels were associated with pregnancy loss in retrospective studies of women with RPL. In this study of women with unexplained RPL, AMH was not associated with the chance of live birth in the next pregnancy. Screening for AMH in women with RPL is not supported by current evidence.
Introduction
Anti-Müllerian hormone (AMH) is a homodimeric glycoprotein produced by the granulosa cells of preantral and antral follicles and is well-established as a measure of the ovarian reserve (Dewailly et al. 2014). Contrary to measures of follicle stimulating hormone and oestradiol, AMH is mainly cycle independent (Fanchin et al. 2003, Hehenkamp et al. 2006, La Marca et al. 2007, van Disseldorp et al. 2010). As such, it is an appealing clinical biomarker to guide fertility treatment and optimize ovarian stimulation protocols (Broer et al. 2011).
Abnormal concentrations of AMH are found in distinct populations of fertility patients (Broekmans et al. 2009). In women with polycystic ovarian syndrome, AMH concentrations are usually high due to the excessive number of small antral follicles (Abbara et al. 2019, Teede et al. 2019). Low concentrations of AMH have been demonstrated in women with premature ovarian failure and in (previous) cancer patients with chemotherapy-induced damage to the ovaries (Knauff et al. 2009, Dewailly et al. 2014).
With increasing age, AMH concentrations decrease, as does the chance of conception per cycle, while pregnancy loss rates increase (Kelsey et al. 2011). At ages 20-24 years, 8.9% of pregnancies were shown to result in a pregnancy loss, while this prevalence rapidly increased after the age of 45 to approximately 74.7% (Nybo Andersen et al. 2000). In patients undergoing in vitro fertilization (IVF) treatments, AMH concentrations have been debated as a prognostic factor for the chance of a live birth (Iliodromiti et al. 2014, Shim et al. 2015, Tal et al. 2015, Gat et al. 2017, Alson et al. 2018, Jiang et al. 2018, Peuranpää et al. 2020). A recent meta-analysis demonstrated an association between low AMH and the risk of pregnancy loss in patients achieving pregnancy by assisted reproductive technology (ART) (Busnelli et al. 2021). Noteworthy, the only prospective study included in the meta-analysis failed to demonstrate such an association (Busnelli et al. 2021). While prognostic outcomes have been thoroughly investigated in infertile women, data is lacking in women with recurrent pregnancy loss (RPL).
RPL affects approximately 2% of all women and in most of these women no explanation can be identified (Stephenson and Kutteh 2007, Ford and Schust 2009, Jaslow et al. 2010). A systematic review demonstrated an increased risk of diminished ovarian reserve (including that of lower AMH concentrations), in 155 women with RPL compared to 158 women without RPL (Bunnewell et al. 2020). In another recent study, the proportion of women with a low AMH increased with an increasing numbers of previous pregnancy losses, also when stratifying results according to the women's age (Tan et al. 2022). However, while there may be an association between AMH concentrations and women diagnosed with RPL, the clinically most important issue is whether there is an association with the risk of yet another pregnancy loss. We hypothesized that AMH concentration was associated with the chance of live birth in the next pregnancy achieved in women with unexplained RPL.
The objective of this study was to investigate the association of AMH with the chance of live birth in a large cohort of women with unexplained RPL.
Materials and Methods
Participants
The present study is a cohort study of all women attending the RPL Unit, a tertiary referral centre with patient uptake from all of Eastern Denmark, at the Copenhagen University Hospital (Rigshospitalet) between June 2015 and December 2021. Women could be referred to the unit if they had had three or more consecutive pregnancy losses or two consecutive late pregnancy losses or stillbirths. As previously described in a study on thyroid peroxidase antibodies (including some of the women included in the present study)(Bliddal et al. 2019), all women underwent extensive examinations upon referral to investigate potential explanations for RPL including antiphospholipid syndrome, severe uterine abnormalities, chromosomal abnormalities, and thyroid autoimmunity or disease (Bliddal et al. 2019). Antiphospholipid syndrome was defined as either positivity for lupus anticoagulant or anticardiolipin antibodies in two separate blood samples drawn at least 12 weeks apart. Thyroid autoimmunity was defined as thyroid peroxidase antibody-positivity (cut-off 60 mIU/L, Kryptor immunofluorescense assay). Both the woman and her male partner were screened for clinically significant chromosomal abnormalities. In the case of women achieving pregnancy by sperm donation, paternal chromosomes were assumed to be normal as karyotype is performed in all sperm donors in Denmark.
Based on these examinations, women with any of the following were excluded: No RPL (<3 previous pregnancy losses); ≥3 previous live births; pregnancies achieved by egg donation; no available AMH measurement; or explained RPL (uterine malformation, clinically significant parental chromosomal abnormality, antiphospholipid syndrome, or thyroid disease including euthyroid women with positivity for thyroid peroxidase antibodies). Further, in the analyses of live birth, pregnancies complicated by risk factors unrelated to RPL were excluded (ectopic pregnancies, twin pregnancies, terminated pregnancies).
All women were offered monitoring of any achieved pregnancy by progesterone and sequential human chorionic gonadotropin measurements every week starting from a positive urine-human chorionic gonadotropin test result, and ultrasound scans at gestational week 6 and every second week thereafter (see Appendix, Figure 1). In case of a suspected pregnancy loss, the loss was confirmed by ultrasound scan(s) and measurements of human chorionic gonadotropin. In case of a viable pregnancy at gestational week 16, the women were referred for further obstetric follow-up at their local hospital for the remainder of the pregnancy. In terms of treatments offered to women with unexplained RPL, intravenous immunoglobulin (Privigen®, CSL Behring GmbH, Germany) was offered if the woman had a minimum of three consecutive unexplained pregnancy losses in pregnancies achieved by IVF or intracytoplasmic sperm injection, in case of secondary RPL with five or more consecutive losses, or to women with a non-thyroidal autoimmune disease (rheumatoid arthritis, Crohn's disease, ulcerative colitis, or myasthenia gravis)) (Christiansen et al. 2015, Egerup et al. 2015, Christiansen et al. 2019). In women achieving pregnancy by IVF or intracytoplasmic sperm injection, prednisolone was given together with intravenous immunoglobulin. Hydroxychloroquine (Plaquenil®, Sanofi, Paris, France) was offered to women with a minimum of four consecutive losses (off-label before 2018 and from 2018 in an ongoing randomized controlled trial). In case of other treatments initiated at fertility clinics such treatment was continued as prescribed by fertility doctors.
Inclusion process of women with unexplained recurrent pregnancy loss followed at the Recurrent Pregnancy Loss Unit between June 2015 and December 2021.
AMH was measured at the Department of Clinical Biochemistry, Copenhagen University Hospital (Rigshospitalet and Hvidovre), by the Roche Elecsys assay on Cobas 8000 (Roche Diagnostics, Basel, Switzerland). The intermediary precision (coefficients of variation) were at 13 pmol/L 7% and at 39 pmol/L, 7%. The analytical sensitivity was 0.07 pmol/L. Local validation established a functional assay sensitivity (corresponding to an intermediary precision of 25%) to 0.21 pmol/L. In categorical analyses, definitions of AMH were based on AMH measurements from women with regular menstrual cycles achieving pregnancy without ART (lower tertile: ≤10 pmol/L, medium tertile: >11 and <21.2 pmol/L, highest tertile: ≥21.2 pmol/L). Although most analyses in the present manuscript were categorical (see statistics section), we chose to include AMH values below 0.21 as absolute values of 0.20 pmol/L in continuous analyses, as we believed the women with very low values represented important biologic information.
Statistical analyses
The primary outcome of this study was live birth in the first pregnancy after referral. Multiple logistic regression analyses were applied to further explore potential predictors of pregnancy and live birth, respectively. Besides AMH group (low, medium, and high) as the main exposure, we included possible confounders: Maternal age at referral (in years), previous number of losses, body mass index (BMI), smoking, and paternal age. In the logistic regression model for chance of pregnancy after referral, irregular menstrual cycle (yes=1, no=0) was also included as covariate, and in the model for live birth, pregnancy by assisted reproductive technology ((ART), in vitro fertilization or intracytoplasmic sperm injection (yes=1, no=0)) and treatment with intravenous immunoglobulin (yes=1, no=0), prednisolone (yes=1, no=0), or hydroxychloroquine (yes=1, no=0) were included. Smoking was defined as ever having smoked (yes=1, no=0) in descriptive AMH analyses and as currently smoking (yes=1, no=0) in analyses of live birth in the first pregnancy after referral. An interaction term between ART and treatment with intravenous immunoglobulin (see treatment indication above) was explored and turned out significant after which it was included in the final logistic regression model. Missing data were not replaced, but differences between subgroups with or without missing data were explored (Appendix, tables A.1-3). Last data extraction was on December 6th corresponding to a minimum follow-up time of one year after referral (median follow-up 47 months, range: 12-107).
Bonferroni correction was applied to adjust for pairwise comparisons of the low/high AMH group to the medium AMH group in the primary analyses (i.e. a p-value of ≤0.025 was considered significant).
Sensitivity analyses were predefined and included other definitions of abnormal AMH; according to the manufacturer's documentation of age-dependent reference ranges, percentile-based cut-offs for 2.5-97.5 in women with regular menstrual cycle and no need for ART, and low AMH defined by 1 ng/mL equivalent to 7.14 pmol/L as used in a few previous studies of AMH. Furthermore, we reran the live birth analysis splitting results in women achieving pregnancy by ART or spontaneously, respectively.
Statistical analyses were performed in SPSS version 25 (IBM, NY, USA). Figures were prepared in Microsoft Excel 2016 (Microsoft, WA, USA) and SPSS version 25 (IBM, NY, USA).
Ethical approval
All data was collected and stored in a secure database with approval from the Danish Data Protection Agency (file number RH-2017-315 I suite 05939). Data was entered by the clinical staff and validated by research associates at the department and, for the purpose of this study, by SB and HSN with a focus on AMH and pregnancy outcome.
Results
A total of 1035 women attended the unit, of which 699 (67.5%) were eligible for inclusion, and of these 629 (90.0%) had a measurement of AMH. Figure 1 is a flowchart of the inclusion process. There were no differences in characteristics between women with and without AMH measurement (Appendix table A.1). Among the 629 women, 222 (35.2%) had previously given live birth, the mean age was 33.5 years (standard deviation (SD), 4.4), and the median number of previous losses was 3 (interquartile range (IQR): 3-4).
AMH concentrations and demographics
Characteristics of the included women according to AMH tertiles are presented in table 1. The median AMH concentration was 16.0 pmol/L (lower tertile 12.0 pmol/L, upper tertile 22.0 pmol/L). All of the 82 (13.1%) women reporting an irregular menstrual cycle had oligomenorrhea, of whom 58 (70.7%) had high AMH, and 18 (22.0%) had medium AMH. Of those reporting of an irregular cycle, six women (7.3%, two with medium AMH and four with high AMH) were taking hormonal contraceptives or metformin at the time of referral controlling their menstrual cycle. Polycystic ovarian syndrome had been previously diagnosed in 18 (2.9%) of the women, of whom 16 had high AMH concentrations.
Table 1Characteristics of women with unexplained recurrent pregnancy loss according to AMH level at referral
In total, 507 (80.6%) women achieved a pregnancy after referral (56.1% within three months, 74% within six months, and all within one year). The proportions were similar between women with low AMH (81.9%), high AMH (80.3%) and medium AMH (79.7%). Women without a pregnancy after referral were older with higher BMI and higher number of previous pregnancy losses, but did not otherwise differ in characteristics including AMH concentrations (see appendix, table A.2). Thus, the adjusted odds of achieving a pregnancy were also similar (low vs medium AMH adjusted odds ratio (aOR) 1.44 95%CI:0.84-2.47, p=0.18, and high vs medium AMH: aOR 0.98, 95%CI:0.59-1.64, p=0.95).
Of the 507 women with a pregnancy after referral, 479 women were eligible for outcome analyses (Figure 1). Among the 28 women excluded from the outcome analyses, fewer had regular menstrual cycles (71.4% vs 88.0%, p=0.02), and more had high AMH (64.3% vs 34.7% in included women, p=0.01), but excluded women did not otherwise differ in demographics (appendix, table A.3). The excluded women with high AMH were mainly excluded due to extrauterine pregnancies (8 extrauterine pregnancies among the 18 excluded with high AMH (44.4%), and 8 (three of which conceived by ART) of 10 extrauterine pregnancies were in women with high AMH (80.0%)).
Live birth according to AMH
The overall live birth rate was 63.7%, and 73.8% of women achieved spontaneous pregnancy. The median number of weeks of gestation in women experiencing pregnancy loss was 6.3 weeks (interquartile range 5.0-8.1 weeks) and in women with live births 39.6 weeks (interquartile range 38.4-40.6).
In the 148 women with a low AMH, live birth rate was 59.5% compared to 66.1% in the 165 women with a medium AMH (OR 0.75 95%CI: 0.48-1.19, p=0.23, aOR with low AMH 0.69, 95%CI:0.42-1.13, p=0.14). The women with low AMH were significantly older than those with medium AMH (mean (SD): 35.0 (4.0) vs 33.2 (4.2) years, p<0.001). Spontaneous conception was achieved by 109 of the 145 (75.2%) women with low AMH (information on ART missing in three women). In the 166 women with a high AMH, live birth rate was 65.1% compared to 66.1% in women with a medium AMH (OR 0.96, 95%CI:0.61-1.51, p=0.85, aOR 0.96, 95%CI:0.59-1.56, p=0.96). Women with a high AMH were younger than women with a medium AMH (mean (SD): 31.4 (4.1) vs 33.2 (4.2) years, p<0.001). Spontaneous conception was achieved by 119 of 164 (72.7%) women with high AMH (information on ART missing for two women).
Neither a low nor a high AMH were associated with live birth in the adjusted analyses (table 2). The chance of live birth was significantly lower in women who had become pregnant by ART (OR 0.57 95%CI: 0.37-0.86, p=0.01, adjusted OR 0.56 95%CI:0.33-0.95, p=0.03), and women with a higher number of previous pregnancy losses (OR 0.87 95%CI:0.76-1.00, p=0.05, adjusted OR 0.80 95%CI:0.68-0.95, p=0.01). Treatment with intravenous immunoglobulin was associated with a higher live birth rate in adjusted analyses (aOR 7.45, 95%CI:1.56-35.53, p=0.01, with a significant interaction term with ART aOR 0.08, 95%:0.01-0.60, p=0.01).
Table 2Live birth in the first pregnancy after referral in women with unexplained recurrent pregnancy loss
Odds ratio for live birth adjusted for covariates: AMH level (low vs medium vs high), age, previous losses, ART treatment, BMI, smoking during pregnancy, treatment with intravenous immunoglobulin, prednisolone, hydroxychloroquine, and paternal age. Missing data from 18 women reduced the adjusted model by this number. The model included an interaction term for ART and intravenous immunoglobulin with aOR 0.08, 95%:0.01-0.60, p=0.013.
Abbreviations: AMH, Anti-Müllerian hormone; ART, assisted reproductive technology; BMI, body mass index; IQR, interquartile range; SD, standard deviation. P-values for the primary outcome of live birth according to AMH category were considered significant if p≤0.025 according to Bonferroni correction.
Odds ratio for live birth adjusted for covariates: AMH level (low vs medium vs high), age, previous losses, ART treatment, BMI, smoking during pregnancy, treatment with intravenous immunoglobulin, prednisolone, hydroxychloroquine, and paternal age. Missing data from 18 women reduced the adjusted model by this number. The model included an interaction term for ART and intravenous immunoglobulin with aOR 0.08, 95%:0.01-0.60, p=0.013.
The decline in AMH according to maternal age was similar in women who had a live birth and those who had another pregnancy loss as illustrated in figure 2A. The odds of live birth were not associated with AMH concentrations (log2 transformed AMH concentration: OR 1.09 95%CI:0.94-1.27, p=0.25, aOR 1.13 95%CI:0.96-1.34, p=0.15). Further, as illustrated by an operator receiving curve, AMH concentrations did not discriminate live birth well (figure 2B).
Figure 2Anti-Müllerian Hormone concentrations and live birth
AMH concentration (log2 transformed) and chance of live birth in the first pregnancy achieved after referral in 473 women with unexplained recurrent pregnancy loss. A, (log2 transformed) AMH concentration according to age at time of referral and outcome of the pregnancy. B, Receiver Operator Curve illustrating the ability of (log2 transformed) AMH concentration to discriminate chance of live birth.
Using the age-dependent reference ranges according to the manufacturer's documentation to define high or low AMH, there was no difference in the chance of live birth in the first pregnancy after referral (live birth with normal AMH 63.8%, low AMH (17 women) 64.7% and high AMH (17 women) 58.8%, p=0.91). As illustrated in figure 2A, even women with the very lowest AMH concentrations did achieve a live birth. Of the 15 women with AMH levels below 1.9 pmol/L (corresponding to below the 2.5th percentile in women with regular cycle and no need for ART), 12 women (30-42 years of age) achieved a first pregnancy after referral (ten out of 12 spontaneously). Excluding one terminated pregnancy, five out of the remaining 11 pregnancies resulted in a live birth (45.5% compared to 64.0% of women with normal AMH, p=0.22). Among the 29 women with AMH concentrations above 56 pmol/L (97.5th percentile in women with regular cycle and no need for ART), 23 achieved pregnancy (16 spontaneously) of whom two had extrauterine pregnancies and 14 out of the remaining 21 had live births (66.7% compared to 64.0% of women with normal AMH, p=0.80). Finally, defining low AMH as below 1 ng/mL equal to 7.14 pmol/L as used in a few previous studies showed no difference in live birth either (56.6% of 76 women with low AMH compared to 65.0% in 403 women with AMH above 1 ng/mL, p=0.19).
Given the finding of significantly lower live birth rates in women achieving pregnancy by ART, analyses were added to explore this. The gestational age at time of pregnancy loss did not differ between ART and non-ART pregnancies (median 6.0, IQR 4.9-7.9 vs. median 6.6, IQR 5.0-8.6). As demonstrated in appendix table A.4, women achieving pregnancy by ART especially differed by less often having given birth previously (primary RPL). However, dividing the adjusted analyses according to previous live birth or not, ART remained significantly associated with a low live birth rate in women without a previous live birth, and borderline significantly in women with a previous live birth (aOR 0.55, 95%CI:0.30-0.99, p=0.045, and aOR 0.37, 95%CI: 0.12-1.15, p=0.09, respectively). Finally, when dividing analyses into women achieving pregnancy spontaneously or by ART, there were still no AMH associations between low or high AMH in either subgroup (appendix, table A.5 and A.6, respectively).
Discussion
In this large cohort study, we investigated characteristics of 629 women with unexplained RPL. We did not find an association between abnormal AMH concentrations and the odds of prospective live birth in the 473 women with a pregnancy after referral. However, women with a higher number of previous pregnancy losses and, especially, women achieving pregnancy by ART, had significantly lower live birth rates, which was somewhat ameliorated in those receiving intravenous immunoglobulin (interaction with ART being significant).
As we hypothesized AMH to be independently associated with live birth, we did not use age-adjusted AMH cut-offs in the primary analyses. However, subgroups of women with RPL and premature ovarian insufficiency or polycystic ovarian syndrome could constitute different phenotypes with a different risk for pregnancy loss (Sjaarda et al. 2018, Mayrhofer et al. 2020). Interestingly, despite our exclusion of all women receiving egg donation, a low AMH concentration was not associated with a reduced chance of achieving pregnancy. In a study of healthy women, Hagen et al found no reduction in pregnancy rate among women with low AMH concentrations (Hagen et al. 2012). Thus, a low AMH outside of an ART setting does not seem to be strongly associated with neither the chance of achieving pregnancy nor of live birth.
A few other studies have investigated the association between AMH measurements and subsequent live birth in women with RPL. In accordance with our findings, Pils et al did not find a lower live birth rate according to AMH concentration in 94 women with unexplained RPL (Pils et al. 2019). The same conclusion was reached by Leclercq et al in a case control study comparing 188 unselected women with RPL to 376 age-matched parous women without pregnancy loss (Leclercq et al. 2019). Finally, two smaller studies also found comparable pregnancy loss rates between women with low and normal AMH (Xiao et al. 2016, McCormack et al. 2019). Contrary to our findings, Murugappan et al did find an association between AMH concentrations and reduced live birth rate in 155 women with RPL (Murugappan et al. 2019). This could be explained by differences in study design such as inclusion of women with uterine factors or antiphospholipid syndrome, multiple pregnancies allowed per woman, and a follow-up period of only 12 months. Notably, low analytical accuracy and precision have challenged comparisons of studies involving AMH assays (Victoria et al. 2019). New automated immunoassays have improved the analytical shortcomings, but the assay used must still be considered a limiting factor in comparison of study results (Nelson et al. 2015, van Helden and Weiskirchen 2015, Victoria et al. 2019).
Women with unexplained RPL constitute a small part of women of reproductive age. Phenotypical variation and differing study designs may explain the conflicting results on the association between AMH concentrations and prospective live birth in women with a healthy reproductive history, with only one or two previous pregnancy losses, or women undergoing ART treatment (Zarek et al. 2016, Tarasconi et al. 2017, Lyttle Schumacher et al. 2018, Hong et al. 2020, Peuranpää et al. 2020). We did not find an association with AMH and live birth, neither when dividing results in women with RPL achieving pregnancy spontaneously nor by ART. Based on our findings, and in accordance with guidelines from the European Society for Human Reproduction and Embryology (Bender Atik et al. 2018), we believe that AMH should not be measured in all women with RPL.
Notably, women achieving pregnancy by ART had odds ratios for live birth of 0.56 compared to those with spontaneous pregnancies. Traditionally, ART has not been considered an independent risk factor in neither women with RPL (Bender Atik et al. 2018) or in women undergoing ART compared to those achieving spontaneous pregnancy (Schieve et al. 2003, Shevell et al. 2005). The women achieving pregnancy by ART were older which could explain some of the increased risk of pregnancy loss. However, the association remained significant also when adjusting for maternal age in the regression model. We did find an increased chance of live birth after treatment with intravenous immunoglobulin (including a significant interaction term between ART and intravenous immunoglobulin). However, confidence intervals were wide, and subgroups were based on a low number of women. Previous studies of intravenous immunoglobulin in the setting of RPL have shown conflicting results (Christiansen et al. 2002, Nyborg et al. 2014, Egerup et al. 2015, Wang et al. 2016, Achilli et al. 2018, Egerup et al. 2022, Yamada et al. 2022). Although the association between ART and low live birth rate in the present study was robust in various sensitivity and subgroup analyses, this finding calls for confirmation in future studies including exploration of specific RPL phenotypes and interventions that could improve prognosis. In an ongoing randomized controlled trial, we are investigating the effect of intravenous immunoglobulin administration in women with RPL after ART (Caroline Nørgaard-Pedersen and Christiansen 2021).
There are strengths and limitations to this study. Especially, the large number of included women with unexplained recurrent pregnancy loss from a large tertiary setting with close follow-up provides important information on prospective live birth after referral. Although there were no differences in characteristics between the women with and without a registered first pregnancy after referral (Appendix, table A.2), some women may have failed to report a pregnancy to the clinic thus inflicting bias. Further, whereas there were no trends towards lower birth rates among those with either low or high AMH concentrations, only five out of ten women with very low AMH concentrations achieved a live birth. Thus, power to detect significant differences in such small subgroups would require very large cohorts or meta-analyses, especially because women with low AMH concentrations were significantly older. Therefore, analyses would need adjustment for maternal age because the lower live birth rate is likely to reflect a negative impact of biological aging (Nelson et al. 2013).
In conclusion, this cohort study of a large number of women with unexplained RPL did not find an association with AMH concentrations and subsequent live birth. AMH should therefore not be included in routine screening of women with RPL. A remarkably lower live birth rate among women with unexplained RPL achieving pregnancy by ART needs to be confirmed and further explored in future studies.
Author contribution
SB and HSN planned the study and drafted the first version of the manuscript. UFR and LH provided expertise on the bioanalytical methodology. JLF supervised the statistical analysis plan. All authors made substantial contributions to the analysis or interpretation of data and the critical revision of the manuscript for important intellectual content. All authors approved the final submitted version.
Funding sources
The research and work related to the preparation of this manuscript has been kindly supported by the Copenhagen University Hospital Rigshospitalet, Musikforlæggerne Agnes and Knut Mørk's Foundation, Desiree and Niels Yde's Foundation, the Danish Medical Association's Research Foundation, the A.P. Møller Foundation for the Advancement of Medical Science, the Lundbeck Foundation, the Danish Thyroid Patient Association, and the Novo Nordisk Foundation. None of the funding sources had any influence on the design and conduct of this study, on the analysis and interpretation of results or on the decision to publish this manuscript.
Data sharing statement
Upon reasonable request, data can be shared according to data protection legislation.
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Data Availability
Data can be made available upon reasonable request and legal permissions obtained
Declaration of Competing Interest
The authors have no conflicts of interest.
Acknowledgments
We owe great thanks to nurses Karen Kirchheiner Jensen, Marie Chonovitsch and Anne-Louise Lunøe for their endless effort in taking care of the many women with RPL followed in our unit. Ulla Feldt-Rasmussen's research salary is supported by a grant from Kirsten and Freddy Johansen's Foundation.
Sofie Bliddal is a medical doctor and earned her PhD degree studying thyroid function in pregnancy. She is currently working as a full-time researcher at the Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark. Her primary research interests are the interplay between hormonal aberrations, immune dysregulation and reproductive failure.
p<0.001 by one-way ANOVA across all three groups
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