Chance of live birth in the first pregnancy after referral among patients with recurrent pregnancy loss is not influenced by their relatives' reproductive history.

Purpose: Recurrent pregnancy loss (RPL) is defined as three or more consecutive pregnancy losses and affects 1-3% of couples trying to conceive. Pregnancy loss is more common among RPL patients' siblings than in the general population. Our objective was to investigate whether first-degree relatives with pregnancy losses influenced the chance of live birth in the first pregnancy after referral among women with RPL.Materials and methods: This is a cohort study of 2138 women with RPL seen at the Danish RPL Unit at Copenhagen University Hospital, Rigshospitalet between January 1st 2000 and December 31st 2017 with follow-up until December 2018. Pregnancies among first-degree relatives were reported by patients at their first consultation. Chance of live birth after referral was compared by logistic regression analysis.Results: Overall, 76% of the referred women achieved a pregnancy after referral and of these, 58% delivered a live born child. Women whose mother had experienced pregnancy loss were referred at a younger age than women with no pregnancy losses among first-degree relatives (mean age 33.6 (SD 4.6) versus 34.3 (SD 4.5), p = 0.002). Pregnancy losses among first-degree relatives did not influence chance of live birth.Conclusions: Our results indicate that pregnancy losses among first-degree family members is not an important risk factor for outcome of the first pregnancy after referral among women with RPL.

Pregnancy outcomes after recurrent pregnancy loss: a longitudinal cohort study on stress and depression.

RESEARCH QUESTION: Are self-reported symptoms of stress and depression associated with pregnancy outcomes within the first year after referral to a tertiary recurrent pregnancy loss unit? DESIGN: Prospective cohort study with online questionnaires using the Major Depression Inventory (MDI) and Cohen's Stress Scale (PSS) at referral and after 1 year. The study was conducted between 2010 and 2014. A total of 301 women who had experienced recurrent pregnancy loss completed the first questionnaire. One year after referral, 185 women (61%) completed a follow-up questionnaire. RESULTS: A score above the threshold for major depression on the MDI at referral was not a predictor for outcome in the first pregnancy after referral; OR (95% CI) for live birth 1.71 (0.66 to 4.44), neither was increasing scores on the PSS: OR 0.98 (95% CI 0.94 to 1.02). At follow-up, women who had achieved a pregnancy resulting in a live birth had significantly lower scores on both the MDI: 13.45 (11.05) versus 11.04 (11.07); difference -2.41 (95% CI -4.60 to -0.23); and the PSS: mean 17.69 (7.59) versus 13.03 (6.83); difference -4.66 (95% CI -6.04 to -3.28), respectively. This was not the case for women who did not have a successful pregnancy. Women who experienced recurrent pregnancy loss after a successful birth were less likely to report symptoms corresponding to major depression than women who had only experienced losses (n = 7 [5%] versus 19 [12%]; P = 0.04). CONCLUSIONS: Self-reported emotional distress did not affect future chance of live birth. A live born child decreased emotional distress.

Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence.

STUDY QUESTION: Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days? SUMMARY ANSWER: Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h. WHAT IS KNOWN ALREADY: Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods. STUDY DESIGN, SIZE, DURATION: This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory's local manual method (Makler chamber) was used for comparison. MAIN RESULTS AND THE ROLE OF CHANCE: The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate. LIMITATIONS, REASONS FOR CAUTION: The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS: Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from 'Ferring Pharmaceuticals' to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Recurrent pregnancy loss: what is the impact of consecutive versus non-consecutive losses?

STUDY QUESTION: Is there a different prognostic impact for consecutive and non-consecutive early pregnancy losses in women with secondary recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Only consecutive early pregnancy losses after the last birth have a statistically significant negative prognostic impact in women with secondary RPL. WHAT IS KNOWN ALREADY: The risk of a new pregnancy loss increases with the number of previous pregnancy losses in patients with RPL. Second trimester losses seem to exhibit a stronger negative impact than early losses. It is unknown whether the sequence of pregnancy losses plays a role for the prognosis in patients with a prior birth. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study of pregnancy outcome in patients with unexplained secondary RPL included in three previously published, Danish double-blinded placebo-controlled trials of intravenous immunoglobulin (IvIg) conducted from 1991 to 2014. No other treatments were given. Patients with documented explained pregnancy losses (ectopic pregnancies and aneuploid miscarriages) were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 168 patients included in the trials, 127 had secondary RPL and experienced a subsequent live birth or unexplained pregnancy loss in the first pregnancy after giving informed consent to participate in the trials (the index pregnancy). Data analyzed by multivariate analysis included the independent variables age, the number of early pregnancy losses before and after the last birth, respectively and a second trimester pregnancy loss before or after the last birth, respectively. The outcome variable was unexplained loss in the index pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: In patients with secondary RPL, both a late and each early loss before the last birth did not significantly influence the risk of a new pregnancy loss in the index pregnancy: incidence rate ratio (IRR) 1.31 (95% CI 0.62-2.77) and IRR 0.88 (95% CI 0.70-1.11), respectively. In contrast, the impact on risk of pregnancy loss conferred by a late and by each early pregnancy loss occurring after the birth was significant: IRR 2.15 (95% CI 1.57-2.94, P < 0.0001) and IRR 1.14 (95% CI 1.04-1.24, P = 0.002), respectively. LIMITATIONS, REASONS FOR CAUTION: Of the patients, 48% were treated with IvIg, which could influence the results. However, allocation to IvIg was random and prognostic variables were equally distributed in IvIg and placebo-treated patients. WIDER IMPLICATIONS OF THE FINDINGS: A birth in women with secondary RPL eradicates the negative prognostic impact of previous pregnancy losses and this finding is important for our understanding of the pathogenesis. It indicates that only consecutive pregnancy losses should count in the definition of RPL. STUDY FUNDING/COMPETING INTERESTS: There was no particular funding for this study. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable for two of the included randomized controlled trials. For the last trial: Clinical.Gov NCT00722475.

Placental magnetic resonance imaging T2* measurements in normal pregnancies and in those complicated by fetal growth restriction.

OBJECTIVES: The magnetic resonance imaging (MRI) variable transverse relaxation time (T2*) depends on multiple factors, one important one being the presence of deoxyhemoglobin. We aimed to describe placental T2* measurements in normal pregnancies and in those with fetal growth restriction (FGR). METHODS: We included 24 normal pregnancies at 24-40 weeks' gestation and four FGR cases with an estimated fetal weight below the 1(st) centile. Prior to MRI, an ultrasound examination, including Doppler flow measurements, was performed. The T2* value was calculated using a gradient echo MRI sequence with readout at 16 different echo times. In normal pregnancies, repeat T2* measurements were performed and interobserver reproducibility was assessed in order to estimate the reproducibility of the method. Placental histological examination was performed in the FGR cases. RESULTS: The method was robust regarding the technical and interobserver reproducibility. However, some slice-to-slice variation existed owing to the heterogeneous nature of the normal placenta. We therefore based T2* estimations on the average of two slices from each placenta. In normal pregnancies, the placental T2* value decreased significantly with increasing gestational age, with mean ± SD values of 120 ± 17 ms at 24 weeks' gestation, 84 ± 16 ms at 32 weeks and 47 ± 17 ms at 40 weeks. Three FGR cases had abnormal Doppler flow, histological signs of maternal hypoperfusion and a reduced T2* value (Z-score < -3.5). In the fourth FGR case, Doppler flow, placental histology and T2* value (Z-score, -0.34) were normal. CONCLUSIONS: The established reference values for placental T2* may be clinically useful, as T2* values were significantly lower in FGR cases with histological signs of maternal hypoperfusion. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications.

Depression and emotional stress is highly prevalent among women with recurrent pregnancy loss.

STUDY QUESTION: Is the prevalence of psychological stress and moderate/severe depression higher for women with recurrent pregnancy loss (RPL) than pregnancy planners trying to conceive naturally? SUMMARY ANSWER: Both psychological stress and major depression are significantly more common among women with RPL than in those trying to conceive naturally. WHAT IS KNOWN ALREADY: RPL has a significant emotional impact on couples, especially the woman. Previous studies have shown inconclusive results. STUDY DESIGN, SIZE, DURATION: In this cross-sectional study, we compared the prevalence of stress and depression among 301 women with RPL and 1813 women attempting to conceive naturally. We defined RPL as three or more pregnancy losses before 12 weeks' gestation. RPL patients were enrolled from 2010 to 2013 and the comparison group from 2011 to 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: RPL patients completed an online questionnaire before their first consultation at the Danish RPL Unit. In addition, we included data from a comparison group of 1813 women who participated in the Soon Parents Study (www.SnartForældre.dk). The Major Depression Index (MDI) was used to assess symptoms of depression, and Cohen's Perceived Stress Scale (PSS) was used to measure stress. Relevant demographic data were also retrieved. MAIN RESULTS AND THE ROLE OF CHANCE: Of the RPL patients, 26 (8.6%) had a score on the MDI corresponding to moderate/severe depression, as did 40 (2.2%) of the women in Soon Parents Study (adjusted odds ratio (OR) 5.53 (95% confidence interval (CI): 2.09; 14.61)). A high stress level, defined as ≥19 on the PSS scale, was reported by 124 (41.2%) of the patients and 420 (23.2%) in the comparison group (adjusted OR 1.59 (95% CI 1.03; 2.44)). LIMITATIONS, REASONS FOR CAUTION: We used online questionnaires, and have no interview data. We were unaware if any of the women in the comparison group suffer from RPL. WIDER IMPLICATIONS OF THE FINDINGS: This study should entail a heightened awareness of mental distress among care providers for women with RPL. STUDY FUNDING/COMPETING INTERESTS: No specific funding was sought for this study. The Soon Parents Study is funded by National Institute of Child Health and Human Development (R01 HD060680-01A4). No authors have competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Terminology for pregnancy loss prior to viability: a consensus statement from the ESHRE early pregnancy special interest group.

Pregnancy loss prior to viability is common and research in the field is extensive. Unfortunately, terminology in the literature is inconsistent. The lack of consensus regarding nomenclature and classification of pregnancy loss prior to viability makes it difficult to compare study results from different centres. In our opinion, terminology and definitions should be based on clinical findings, and when possible, transvaginal ultrasound. With this Early Pregnancy Consensus Statement, it is our goal to provide clear and consistent terminology for pregnancy loss prior to viability.

Intravenous immunoglobulin treatment for secondary recurrent miscarriage: a randomised, double-blind, placebo-controlled trial.

OBJECTIVE: To determine whether infusions with intravenous immunoglobulin (IVIg) during early pregnancy increase live birth rate in women with secondary recurrent miscarriage compared with placebo. DESIGN: A single-centre, randomised, double-blind, placebo-controlled trial. SETTING: A tertiary centre for recurrent miscarriage in Copenhagen, Denmark. POPULATION: A group of 82 women with unexplained secondary recurrent miscarriage and at least four miscarriages. METHODS: Women were randomly assigned to repeated infusions with IVIg or placebo (albumin) from the time of positive pregnancy test to gestational week 15 or pregnancy loss. MAIN OUTCOME MEASURE: Primary outcome was birth with neonatal survival in all randomised women. RESULTS: In the intention-to-treat analyses, live birth rates were 23/42 (54.8%) in the IVIg and 20/40 (50.0%) in the placebo group, relative risk 1.11 (95% CI 0.70-1.74). In a per protocol analysis, almost identical results were found. The median gestational length at delivery was higher in the IVIg than the placebo group (282 versus 272 days, P = 0.02) but the mean birthweight was not significantly increased. CONCLUSIONS: In this trial, which is the largest so far, IVIg did not increase the live birth rate in patients with secondary recurrent miscarriage and the treatment cannot be recommended in clinical practice.

Non-visualized pregnancy losses are prognostically important for unexplained recurrent miscarriage.

STUDY QUESTION: Are non-visualized pregnancy losses (biochemical pregnancy loss and failed pregnancy of unknown location combined) in the reproductive history of women with unexplained recurrent miscarriage (RM) negatively associated with the chance of live birth in a subsequent pregnancy? SUMMARY ANSWER: Non-visualized pregnancy losses contribute negatively to the chance for live birth: each non-visualized pregnancy loss confers a relative risk (RR) for live birth of 0.90 (95% CI 0.83; 0.97), equivalent to the RR conferred by each additional clinical miscarriage. WHAT IS KNOWN ALREADY: The number of clinical miscarriages prior to referral is an important determinant for live birth in women with RM, whereas the significance of non-visualized pregnancy losses is unknown. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study comprising 587 women with RM seen in a tertiary RM unit 2000-2010. Data on the outcome of the first pregnancy after referral were analysed for 499 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in the RM Unit at Rigshospitalet, Copenhagen, Denmark. We included all women with unexplained RM, defined as ≥3 consecutive clinical miscarriages or non-visualized pregnancy losses following spontaneous conception or homologous insemination. The category 'non-visualized pregnancy losses' combines biochemical pregnancy loss (positive hCG, no ultrasound performed) and failed PUL (pregnancy of unknown location, positive hCG, but on ultrasound, no pregnancy location established). Demographics were collected, including BMI, age at first pregnancy after referral and outcome of pregnancies prior to referral. Using our own records and records from other Danish hospitals, we verified the outcome of the first pregnancy after referral. For each non-visualized pregnancy loss and miscarriage in the women's reproductive history, the RR for live birth in the first pregnancy after referral was determined by robust Poisson regression analysis, adjusting for risk factors for negative pregnancy outcome. MAIN RESULTS AND THE ROLE OF CHANCE: Non-visualized pregnancy losses constituted 37% of reported pregnancies prior to referral among women with RM. Each additional non-visualized pregnancy loss conferred an RR for live birth of 0.90 (95% CI 0.83; 0.97), which was not statistically significantly different from the corresponding RR of 0.87 (95% CI 0.80; 0.94) conferred by each clinical miscarriage. Among women with ≥2 clinical miscarriages, a reduced RR for live birth was also shown: 0.82 (95% CI 0.74; 0.92) for each clinical miscarriage and 0.89 (95% CI 0.80; 0.98) for each non-visualized pregnancy loss, respectively. Surgically treated ectopic pregnancies (EPs) were significantly more common for women with primary RM and no confirmed clinical miscarriages, compared with women with primary RM and ≥1 clinical miscarriage (22 versus 6%, difference 16% (95% CI 9.1%; 28.7%); RR for ectopic pregnancy was 4.0 (95% CI 1.92; 8.20). LIMITATIONS, REASONS FOR CAUTION: RM was defined as ≥3 consecutive pregnancy losses before 12 weeks' gestation, and we included only women with unexplained RM after thorough evaluation. It is uncertain whether the findings apply to other definitions of RM and among women with known causes for their miscarriages. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first comprehensive investigation of prior non-visualized pregnancy losses and their prognostic significance for live birth in a subsequent pregnancy in women with unexplained RM. We show that a prior non-visualized pregnancy loss has a negative prognostic impact on subsequent live birth and is thus clinically significant. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Angiogenic factors and the lectin pathway of complement in women with secondary recurrent pregnancy loss.

The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.

NLRP7 or KHDC3L genes and the etiology of molar pregnancies and recurrent miscarriage.

Women with mutation in both alleles of the NLRP7 or C6orf221/KHDC3L genes are predisposed to diploid biparental moles, but it has also been suggested that mutation in these genes can predispose to diploid androgenetic or triploid moles and to other kinds of reproductive wastage. We have investigated the association between molar pregnancy and recurrent miscarriages regarding changes in the NLRP7 and C6orf221/KHDC3L genes. Our study group can be divided into three sub-cohorts: (i) women having had at least one molar pregnancy and at least two non-mole miscarriages, (ii) women having had recurrent androgenetic hydatidiform moles and (iii) women having had one diploid androgenetic hydatidiform mole and a relative having had a hydatidiform mole (familial hydatidiform moles). We observed a statistically non-significant tendency of non-synonymous variants in NLRP7 to be more frequent in women with familial hydatidiform mole and in women with female family members with hydatidiform mole or non-mole miscarriage compared with women with no family history of mole or miscarriage. However, we did not find any unequivocal pathogenic mutations (the term 'unequivocal pathogenic mutations' refers to mutations that indubitably have a pathogenic effect on the affected woman) in NLRP7 or C6orf221/KHDC3L in any of the women in the study group. This indicates that recurrent miscarriages plus hydatidiform mole, recurrent androgenetic hydatidiform moles and familial androgenetic hydatidiform moles in general do not have the same monogenetic etiology as familiar diploid biparental moles.

Maternal carriage of H-Y restricting HLA class II alleles is a negative prognostic factor for women with recurrent pregnancy loss after birth of a boy.

Immune system aberrations are suggested to be an important factor in the pathophysiology of unexplained secondary recurrent pregnancy loss (sRPL). The objective was to investigate if the sex ratio of the firstborn child in sRPL patients differs from the background population and whether the sex of the firstborn child has a negative impact on the pregnancy prognosis alone and/or in combination with carriage of male-specific minor histocompatibility (H-Y) restricting HLA class II alleles. From January 2016 to October 2022, 582 patients with unexplained RPL were admitted to the RPL Center of Western Denmark and continuously followed-up. HLA-DRB1 and -DQB1 typing was performed as part of the routine diagnostic work-up. In sRPL patients, a history of a firstborn boy was significantly more frequent than in the Danish background population and was associated with significantly lower odds of a successful reproductive outcome in the first pregnancy after admission compared to a firstborn girl (OR=0.41, 95% CI: 0.20-0.83, p = 0.014). The odds of a successful reproductive outcome were enhanced in patients carrying ≥ 1 H-Y-restricting HLA class II alleles with a first-born girl compared to a firstborn boy (OR=3.33, 95% CI: 1.40-7.88, p = 0.005), while no difference in successful reproductive outcome was seen in sRPL patients not carrying these alleles (OR=1.20, 95% CI: 0.33-4.43, p = 0.781). The sex ratio of children born after RPL was similar to the Danish background population. These findings confirm previous findings and suggests that a harmful immune response triggered by H-Y-antigen exposure during a previous pregnancy in preconditioned women may cause sRPL.

Plasma level of mannose-binding lectin is associated with the risk of recurrent pregnancy loss but not pregnancy outcome after the diagnosis.

STUDY QUESTION: Are low or high plasma mannose-binding lectin (p-MBL) levels associated with recurrent pregnancy loss (RPL) and the reproductive and perinatal outcomes before and after RPL? SUMMARY ANSWER: The prevalence of low p-MBL levels was significantly higher in RPL patients, while high levels were significantly less prevalent. No association was found between p-MBL level and reproductive and perinatal outcomes before and after RPL. WHAT IS KNOWN ALREADY: Mannose-binding lectin (MBL) is an important component in the innate immune system. Low p-MBL levels have been associated with RPL, while the correlation with high levels has been poorly studied. Adverse perinatal outcomes are generally more frequent among RPL patients, but reports concerning the association between maternal p-MBL levels and perinatal outcomes, including birth weight (BW) and gestational age (GA), are conflicting. STUDY DESIGN SIZE DURATION: This study was a combined cross-sectional and cohort study of 267 RPL patients admitted to the RPL Center of Western Denmark between January 2016 and March 2020. RPL patients were followed until birth of a liveborn child or until end of follow-up, March 2021. A sample of 185 healthy female blood donors of reproductive age was used as a MBL reference group. PARTICIPANTS/MATERIALS SETTING METHODS: All RPL patients had ≥3 consecutive pregnancy losses, a regular menstrual cycle and no known significant chromosomal or uterine malformations. At the first consultation, routine blood samples including p-MBL measurement and detailed obstetrical and perinatal information were collected. p-MBL levels in RPL patients were compared to the MBL reference group. A logistic regression analysis adjusted for relevant confounders assessed the association between low p-MBL levels and an unsuccessful reproductive outcome in RPL patients in first pregnancy after admission. Perinatal outcomes before and after RPL were compared between RPL subgroups according to low (≤500 µg/l), intermediate (501-3000 µg/l) and high (>3000 µg/l) p-MBL levels. MAIN RESULTS AND THE ROLE OF CHANCE: Significantly more RPL patients had low p-MBL levels (prevalence proportion ratio (PPR): 1.79, 95% CI: 1.34-2.38) and fewer had high p-MBL levels (PPR: 0.56, 95% CI: 0.40-0.79) compared to the reference group, while the prevalence of intermediate p-MBL level was not different between the groups (PPR: 0.86, 95% CI: 0.69-1.08). In the prospective study, low p-MBL level was not a significant risk factor for a pregnancy loss in the first pregnancy after admission after adjustment for age, BMI and smoking. Neither before nor after the RPL diagnosis were maternal p-MBL levels significantly associated with BW or GA. LIMITATIONS REASONS FOR CAUTION: Only 161 (60.3%) patients had given birth after RPL during the follow-up period, which limited the possibility to detect clear associations between p-MBL levels and perinatal outcomes after RPL. WIDER IMPLICATIONS OF THE FINDINGS: In agreement with several previous studies, low p-MBL levels are strongly associated with RPL, while this study for the first time documents that high levels may play a protective role, which suggests a causal relationship. We suggest that larger prospective studies evaluate the association between p-MBL levels and RPL prognosis. STUDY FUNDING/COMPETING INTERESTS: No external funding was received. We acknowledge the Department of Obstetrics and Gynaecology at Aalborg University Hospital for financial support. U.S.K. has reported personal fees from Merck, consulting fees from IBSA Nordic, and a grant from Gedeon Richter, Merck and IBSA Nordic outside of the submitted work. TRIAL REGISTRATION NUMBER: ID from clinicaltrials.gov is NCT04017754.

A genome-wide scan in affected sibling pairs with idiopathic recurrent miscarriage suggests genetic linkage.

Previously, siblings of patients with idiopathic recurrent miscarriage (IRM) have been shown to have a higher risk of miscarriage. This study comprises two parts: (i) an epidemiological part, in which we introduce data on the frequency of miscarriage among 268 siblings of 244 patients with IRM and (ii) a genetic part presenting data from a genome-wide linkage study of 38 affected sibling pairs with IRM. All IRM patients (probands) had experienced three or more miscarriages and affected siblings two or more miscarriages. The sibling pairs were genotyped by the Affymetrix GeneChip 50K XbaI platform and non-parametric linkage analysis was performed via the software package Merlin. We find that siblings of IRM patients exhibit a higher frequency of miscarriage than population controls regardless of age at the time of pregnancy. We identify chromosomal regions with LOD scores between 2.5 and 3.0 in subgroups of affected sibling pairs. Maximum LOD scores were identified in four occurrences: for rs10514716 (3p14.2) when analyzing sister-pairs only; for rs10511668 (9p22.1) and rs341048 (11q13.4) when only analyzing families where the probands have had four or more miscarriages; and for rs10485275 (6q16.3) when analyzing one sibling pair from each family only. We identify no founder mutations. Concluding, our results imply that IRM patients and their siblings share factors which increase the risk of miscarriage. In this first genome-wide linkage study of affected sibling pairs with IRM, we identify regions on chromosomes 3, 6, 9 and 11 which warrant further investigation in order to elucidate their putative roles in the genesis of IRM.

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 h) sexual abstinence periods.

OBJECTIVE: Metabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described. To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men. STUDY DESIGN: Semen samples collected after long (4-7 days) and short abstinence (2 h) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy. RESULTS: In total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume × concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12-3.87) vs. 1.29 (0.83-2.62) (P < 0.001) and taurine 0.58 (0.36-0.92) vs. 0.43 (0.28-0.95) (P < 0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence. CONCLUSION: The generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples. The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

HLA-DRB1 polymorphism in recurrent pregnancy loss: New evidence for an association to HLA-DRB1*07.

Many cases of recurrent pregnancy loss (RPL) defined as ≥3 consecutive pregnancy losses are suggested to be caused by an aberrant maternal immune response against the fetus or trophoblast. Human leukocyte antigen (HLA)-DRB1 and -DQB1 polymorphisms are associated with most autoimmune disorders and studies of HLA-DBB1 polymorphism in RPL patients are thus relevant. In previous studies, the HLA-DRB1*03 allele was found with increased prevalence in RPL patients. We wanted to clarify whether HLA-DRB1 alleles indeed were associated with RPL among women of Caucasian descent. A total of 1078 women with unexplained RPL and 2066 bone marrow donors were HLA-DRB1-typed and subsets were also HLA-DQB1 typed. All patients were initially HLA-DRB1-typed by DNA-based low-resolution techniques and subsets of patients and all controls were typed by high-resolution techniques. Among patients, the HLA-DRB1*07 allele frequency was significantly increased compared with controls; OR 1.29 (95 % CI 1.09-1.52), p < 0.0025; after correction for multiple comparisons pc = 0.031. The HLA-DRB1*07/*07 genotype was highly increased in patients with RPL compared with controls: OR 2.27 (1.31-3.93), p = 0.0027. The frequency of the HLA-DRB1*07 phenotype in RPL patients had increased significantly (p = 0.002) in three studies from our group published 1994-2021. The allele frequency of HLA-DRB1*03 was not increased in RPL patients compared with controls; OR 0.96 (0.83-1.12). In conclusion, the previous association between HLA-DRB1*03 and RPL could not be confirmed in our study whereas an association to HLA-DRB1*07 was detected for the first time. Since the latter association is a new finding, it should be confirmed in future studies.

Hereditary thrombophilia and recurrent pregnancy loss: a retrospective cohort study of pregnancy outcome and obstetric complications.

BACKGROUND: The association among hereditary thrombophilia, recurrent pregnancy loss (RPL) and obstetric complications is yet uncertain. The objective of the study was to assess the prognostic value of the factor V Leiden (FVL) and prothrombin (PT) mutations for the subsequent chance of live birth for women with RPL. METHODS: Pregnancy outcome was recorded in a retrospective cohort of 363 women with a minimum of three consecutive pregnancy losses (early miscarriage, late miscarriage or stillbirth/neonatal death) who were not treated with anticoagulation therapy. RESULTS: Of the 363 women, 29 were FVL-mutation carriers and 6 were PT-mutation carriers. The unadjusted live birth rate was 45.7% in FVL/PT carriers versus 63.4% in FVL/PT non-carriers, P = 0.04. The adjusted odds ratio for live birth in FVL/PT carriers was 0.48 (95% CI = 0.23-1.01), P = 0.05. Among the obstetric complications, only excessive bleeding was found to be associated with FVL/PT mutations. CONCLUSIONS: In the unadjusted analysis, FVL and PT mutations have a negative prognostic impact on the live birth rate in women with RPL; however, when adjusting for significant covariates, the results no longer reach statistical significance. Strong conclusions on the association between obstetric complications and hereditary thrombophilia cannot be drawn from this study. Whether anticoagulation therapy would improve the prognosis in women with RPL and FVL/PT mutations remains to be documented in large randomized controlled trials.

Frequency and impact of obstetric complications prior and subsequent to unexplained secondary recurrent miscarriage.

BACKGROUND: The chance of a live birth after a diagnosis of secondary recurrent miscarriage (SRM) is reduced in patients who, prior to the miscarriages, gave birth to a boy and carry HLA class II alleles that efficiently present male-specific (H-Y) antigens to the immune system. Information about obstetric complications in births prior and subsequent to the SRM diagnosis is limited. The relations between maternal carriage of H-Y-restricting HLA, fetal sex, obstetric complications and prognosis are unknown. METHODS: Women with unexplained SRM referred to the Danish Recurrent Miscarriage Clinic between 1986 and 2006 (n = 358) were included; 213 gave birth after the diagnosis. Controls, retrieved from the Danish National Birth Registry, were all women with singleton birth of parity 0, 1982-2005 (n = 608,068) and parity 1, 1986-2008 (n = 510,264). Cross-linkage to the National Discharge Registry identified birth complication diagnoses related to the relevant births among patients and controls. RESULTS: The sex ratio was 1.49 in births prior to SRM and 0.76 in birth after SRM (P < 0.0001). For SRM patients with only late miscarriages (>10 weeks gestation), the corresponding sex ratios were 2.31 and 0.21. Compared with the control groups, obstetric complications were more frequent both before (39% versus 24% P

H-Y antibody titers are increased in unexplained secondary recurrent miscarriage patients and associated with low male : female ratio in subsequent live births.

BACKGROUND: The birth of a boy is significantly more common than a girl prior to secondary recurrent miscarriage (SRM) and is associated with a poorer chance of a subsequent live birth. Children born after SRM are more likely to be girls. High-titer antisera specific for male antigens (H-Y) have been shown to arrest development of male bovine embryos efficiently. We consequently questioned the role of H-Y antibodies in women with SRM. METHODS: Serum samples from patients with unexplained SRM (n = 84), unexplained primary recurrent miscarriage (PRM) (n = 12) and healthy women (n = 37) were obtained. The samples were taken during pregnancy (gestational weeks 4-5) for 77 (80%) of the patients. Enzyme-linked immunosorbent assay was used to detect immunoglobulin G antibodies that specifically recognized any of the five recombinant H-Y proteins (EIF1AY, RPS4Y1, ZFY, DDX3Y and UTY) and their H-X homologs. RESULTS: H-Y-specific antibodies were more frequent in SRM patients (46%) compared with female controls (19%, P = 0.004) and PRM patients (8%, P = 0.01). The presence of H-Y antibodies in early pregnancy was associated with a low male: female birth ratio among the subsequent live births, as only 12% of children born to H-Y antibody-positive patients were boys compared with 44% boys born to H-Y antibody negative patients (P = 0.03). CONCLUSIONS: The high frequency of H-Y antibody-positive SRM patients and the association between the presence of these antibodies in early pregnancy and the low number of male offspring, suggest that maternal immune responses against H-Y antigens can cause pregnancy losses. Further exploring these mechanisms may increase our understanding of unexplained SRM.