The development of an indel panel for microchimerism detection.

OBJECTIVES: The aim of the study was to create a simple assay for microchimerism detection independent of sex and without HLA genotyping. METHODS: The method is based on detection of insertion or deletions utilizing a multiplex PCR followed by fragment analysis by capillary electrophoresis, and probe-based qPCR assays. A total of 192 samples, taken either before pregnancy, during 1st trimester, or either during 2nd trimester or at miscarriage, obtained from a cohort of 97 female patients with either primary or secondary recurrent pregnancy loss, were screened for fetal microchimerism by the indel panel as well as an existing assay based on detection of the Y-chromosome marker; DYS14. RESULTS: The overall prevalence of DYS14 positive samples was 29% (55/192) whereas 32% (61/192) tested positive by the indel method. There was an overall agreement of 64% (122/192) between the results obtained by the two methods. A Fisher's Exact test showed no statistic significant difference in the prevalence of microchimerism detected by the two methods at any of the three times of sampling. The distribution of the number of positive wells detected by both methods were compared by a Mann-Whitney U test, which showed no statistically significant difference at any of the three times of sampling. CONCLUSION: The data indicates that microchimerism can be detected efficiently by the indel method. This makes it possible to detect both female and male cells without the need of HLA-genotyping. Furthermore, the indel method has potential to be implemented as a routine analysis. This will remove the sex bias in future explorations of the role microchimerism plays in health and disease.

The impact of early pregnancy complications on completed family size-A nationwide, registry-based cohort study with 40 years of data.

INTRODUCTION: The impact of early pregnancy complications on completed family size is unknown. Here, we hypothesize that early pregnancy complications and adverse outcomes may influence family size. MATERIAL AND METHODS: In this nationwide, registry-based study we included all 458 475 women born 1957-1972 who lived in Denmark from age 20-45 years with at least one registered pregnancy. The main outcome of the study was number of children per woman by age 45, estimated using a Generalized Linear Mixed Model. Exposures were: (a) total number of pregnancy losses experienced (0, 1, 2, ≥3); (b) highest number of consecutive pregnancy losses (0, 1, 2, ≥3); (c) sex of firstborn child; (d) outcome of first pregnancy (live birth, stillbirth, pregnancy loss, ectopic pregnancy, or molar pregnancy). RESULTS: Number of live births was negatively influenced by maternal age and adverse first pregnancy outcomes, especially ectopic pregnancies. A 30-year-old woman with a first ectopic pregnancy was expected to have 1.16 children (95% CI 1.11-1.22) compared with 1.95 children (95% CI 1.86-2.03) with a first live birth. Three or more consecutive losses also decreased number of live births significantly: 1.57 (95% CI 1.50-1.65) compared with 1.92 (95% CI 1.84-2.0) with only live births. The total number of pregnancy losses had no effect before the age of 35 years. Sex of firstborn had no effect. CONCLUSIONS: Previous pregnancy history has a significant effect on number of children per woman, which is important at both individual and societal levels. Pathophysiological research of adverse pregnancy outcomes should be an urgent priority as the causes remain poorly understood.

Chance of live birth: a nationwide, registry-based cohort study.

STUDY QUESTION: Does the sequence of prior pregnancy events (pregnancy losses, live births, ectopic pregnancies, molar pregnancy and still birth), obstetric complications and maternal age affect chance of live birth in the next pregnancy and are prior events predictive for the outcome? SUMMARY ANSWER: The sequence of pregnancy outcomes is significantly associated with chance of live birth; however, pregnancy history and age are insufficient to predict the outcome of an individual woman's next pregnancy. WHAT IS KNOWN ALREADY: Adverse pregnancy outcomes decrease the chance of live birth in the next pregnancy, whereas the impact of prior live births is less clear. STUDY DESIGN, SIZE, DURATION: Nationwide, registry-based cohort study of 1 285 230 women with a total of 2 722 441 pregnancies from 1977 to 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women living in Denmark in the study period with at least one pregnancy in either the Danish Medical Birth Registry or the Danish National Patient Registry. Data were analysed using logistic regression with a robust covariance model to account for women with more than one pregnancy. Model discrimination and calibration were ascertained using 20% of the women in the cohort randomly selected as an internal validation set. MAIN RESULTS AND THE ROLE OF CHANCE: Obstetric complications, still birth, ectopic pregnancies and pregnancy losses had a negative effect on the chance of live birth in the next pregnancy. Consecutive, identical pregnancy outcomes (pregnancy losses, live births or ectopic pregnancies) immediately preceding the next pregnancy had a larger impact than the total number of any outcome. Model discrimination was modest (C-index = 0.60, positive predictive value = 0.45), but the models were well calibrated. LIMITATIONS, REASONS FOR CAUTION: While prior pregnancy outcomes and their sequence significantly influenced the chance of live birth, the discriminative abilities of the predictive models demonstrate clearly that pregnancy history and maternal age are insufficient to reliably predict the outcome of a given pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: Prior pregnancy history has a significant impact on the chance of live birth in the next pregnancy. However, the results emphasize that only taking age and number of losses into account does not predict if a pregnancy will end as a live birth or not. A better understanding of biological determinants for pregnancy outcomes is urgently needed. STUDY FUNDING/COMPETING INTEREST(S): The work was supported by the Novo Nordisk Foundation, Ole Kirk Foundation and Rigshospitalet's Research Foundation. The authors have no financial relationships that could appear to have influenced the work. TRIAL REGISTRATION NUMBER: N/A.

Pregnancy loss: A 40-year nationwide assessment.

INTRODUCTION: Pregnancy loss is frequent. We aimed to assess the frequency and trends in pregnancy losses according to female age and mode of conception over a 40-year follow-up period. MATERIAL AND METHODS: In a national historical prospective cohort study, we followed all Danish women 10-49 years over the 40-year study period 1978-2017. Data on pregnancies and their outcomes were obtained from the National Health Registry, the Medical Birth Registry and the National Fertility Registry. Incidence rates per 100 pregnancies and per 1,000 women-years as well as lifetime risks per 100 women were calculated. Women included in the lifetime analysis were followed from age 12 to age 49. Pregnancy loss included spontaneous abortion, missed abortion and anembryonic pregnancy. RESULTS: In 3 519 455 recorded pregnancies, 337 008, or 9.6%, were diagnosed with a pregnancy loss. The proportion increased from 7.5% in 1978-1979, peaked at 10.7% in 2000 and thereafter decreased to 9.1% in 2015-2017. Pregnancy loss rate in women 10-14 years was 3.9%, increasing gradually with age to 26.9% in pregnant women 45-49 years, a 6.9-fold increase. Loss rates were slightly lower in naturally conceived pregnancies than in assisted pregnancies except for women above 45 years, where the risk of loss was higher in the spontaneously conceived group. Lifetime risk of specific numbers of losses were: 0: 76.9%, 1: 17.9%, 2: 3.9%, 3: 0.87%, and 4+: 0.35%. CONCLUSIONS: The proportion of women experiencing pregnancy loss has changed little throughout four decades and is still primarily influenced by female age. More than 75% of pregnant women are never recorded with a pregnancy loss, and <1.5% will experience three or more losses.

Recurrent pregnancy loss: diagnostic workup after two or three pregnancy losses? A systematic review of the literature and meta-analysis.

BACKGROUND: Recurrent pregnancy loss (RPL) occurs in 1-3% of all couples trying to conceive. No consensus exists regarding when to perform testing for risk factors in couples with RPL. Some guidelines recommend testing if a patient has had two pregnancy losses whereas others advise to test after three losses. OBJECTIVE AND RATIONALE: The aim of this systematic review was to evaluate the current evidence on the prevalence of abnormal test results for RPL amongst patients with two versus three or more pregnancy losses. We also aimed to contribute to the debate regarding whether the investigations for RPL should take place after two or three or more pregnancy losses. SEARCH METHODS: Relevant studies were identified by a systematic search in OVID Medline and EMBASE from inception to March 2019. A search for RPL was combined with a broad search for terms indicative of number of pregnancy losses, screening/testing for pregnancy loss or the prevalence of known risk factors. Meta-analyses were performed in case of adequate clinical and statistical homogeneity. The quality of the studies was assessed using the Newcastle-Ottawa scale. OUTCOMES: From a total of 1985 identified publications, 21 were included in this systematic review and 19 were suitable for meta-analyses. For uterine abnormalities (seven studies, odds ratio (OR) 1.00, 95% CI 0.79-1.27, I2 = 0%) and for antiphospholipid syndrome (three studies, OR 1.04, 95% CI 0.86-1.25, I2 = 0%) we found low quality evidence for a lack of a difference in prevalence of abnormal test results between couples with two versus three or more pregnancy losses. We found insufficient evidence of a difference in prevalence of abnormal test results between couples with two versus three or more pregnancy losses for chromosomal abnormalities (10 studies, OR 0.78, 95% CI 0.55-1.10), inherited thrombophilia (five studies) and thyroid disorders (two studies, OR 0.52, 95% CI: 0.06-4.56). WIDER IMPLICATIONS: A difference in prevalence in uterine abnormalities and antiphospholipid syndrome is unlikely in women with two versus three pregnancy losses. We cannot exclude a difference in prevalence of chromosomal abnormalities, inherited thrombophilia and thyroid disorders following testing after two versus three pregnancy losses. The results of this systematic review may support investigations after two pregnancy losses in couples with RPL, but it should be stressed that additional studies of the prognostic value of test results used in the RPL population are urgently needed. An evidenced-based treatment is not currently available in the majority of cases when abnormal test results are present.

Pregnancy Loss and Cancer Risk: A Nationwide Observational Study.

BACKGROUND: Cancer is the second leading cause of death worldwide. Few studies have investigated if recurrent pregnancy loss is associated with an increased risk of cancer. We aimed to assess whether pregnancy loss is associated with later cancer development. METHODS: We identified all invasive cancers after age 40, among all Danish women born between January 1957 and December 1972, ensuring a full reproductive history. Cases were matched by birth year 1:10 to cancer-free controls. Women were followed until the end of 2017. The number of pregnancy losses (miscarriages or still births) was correlated to long-term cancer risk using conditional logistic regression, providing odds ratios for specific cancers with different numbers of pregnancy losses, all adjusted for age, education, and other potential confounders. FINDINGS: The study included 28,785 women with cancer (mean age 48.7 [SD 5.0]) and 283,294 matched controls (mean age 48.6 [SD 5.0]). We found no overall association between pregnancy loss and later development of 11 site-specific types of cancer or cancer overall. Taking the sequence of pregnancy losses into account, primary recurrent pregnancy loss (three consecutive pregnancy losses without prior live birth) was associated with later overall cancer by an odds ratio of 1.27 (1.04-1.56). Secondary recurrent pregnancy loss showed no association to cancer. INTERPRETATION: Pregnancy loss was not associated with later cancer development. Women with primary recurrent pregnancy loss had a borderline significant association to later cancer overall, this may be a chance finding. FUNDING: Ole Kirk's Foundation and Copenhagen University Hospital Rigshospitalet's Research Grant.

Treatment with intravenous immunoglobulin in patients with recurrent pregnancy loss: An update.

Intravenous immunoglobulin (IVIg) has a documented clinical effect in many autoimmune diseases and has so far been tested in >10 randomised controlled trials (RCTs) in women with recurrent pregnancy loss (RPL). The results of the RCTs have, however, been very divergent. In meta-analyses of all trials, no significant impact on live birth rate has been reported. In contrast, in sensitivity analyses, IVIg significantly increased live birth rates when initiated prior to conception and it had a borderline significant therapeutic effect in women with secondary RPL. Higher dosages of IVIg and serological signs of autoimmunity in the treated patients tended to increase the success rate after treatment. A follow-up study of patients from our recent RCT also supports a significant therapeutic effect in patients who had received IVIg before conception. The lessons learned from the published trials and meta-analyses should be incorporated in the design of future RCTs of IVIg in the treatment of RPL.

Reproductive Endocrinology in Recurrent Pregnancy Loss.

Endocrine disruptions may be important in patients experiencing recurrent pregnancy loss (RPL). This review focuses on data available on RPL and the endocrine system to investigate relevant, and perhaps modifiable, endocrine factors of importance for the disorder. Evidence indicates that some hormones may be important as immune modulators and a better understanding of this interplay has potential for improving pregnancy outcome in RPL. To date there is a lack of consensus on the effect of endocrine treatment options in RPL and there is a strong need for large randomized-controlled trials.

Immunologic Abnormalities, Treatments, and Recurrent Pregnancy Loss: What Is Real and What Is Not?

Recurrent pregnancy loss, depending on the definition, affects 1% to 3% of women aiming to have a child. Little is known about the direct causes of recurrent pregnancy loss, and the condition is considered to have a multifactorial and complex pathogenesis. The aim of this review was to summarize the evaluation and the management of the condition with specific emphasis on immunologic biomarkers identified as risk factors as well as current immunologic treatment options. The review also highlights and discusses areas in need of further research.

Inheritance of the 8.1 ancestral haplotype in recurrent pregnancy loss.

BACKGROUND AND OBJECTIVES: The 8.1 ancestral haplotype (AH) (HLA-A1, C7, B8, C4AQ0, C4B1, DR3, DQ2) is a remarkably long and conserved haplotype in the human major histocompatibility complex. It has been associated with both beneficial and detrimental effects, consistent with antagonistic pleiotropy. It has also been proposed that the survival of long, conserved haplotypes may be due to gestational drive, i.e. selective miscarriage of fetuses who have not inherited the haplotype from a heterozygous mother. Recurrent pregnancy loss (RPL) is defined as three or more consecutive pregnancy losses. The objective was to test the gestational drive theory for the 8.1AH in women with RPL and their live born children. METHODOLOGY: We investigated the inheritance of the 8.1AH from 82 heterozygous RPL women to 110 live born children. All participants were genotyped for HLA-A, -B and -DRB1 in DNA from EDTA-treated blood or buccal swaps. Inheritance was compared with a Mendelian inheritance of 50% using a two-sided exact binomial test. RESULTS: We found that 55% of the live born children had inherited the 8.1AH, which was not significantly higher than the expected 50% (P = 0.29). Interestingly, we found a non-significant trend toward a higher inheritance of the 8.1AH in girls, 63%, P = 0.11 as opposed to boys, 50%, P = 1.00. CONCLUSIONS AND IMPLICATIONS: We did not find that the 8.1AH was significantly more often inherited by live born children of 8.1AH heterozygous RPL women. However our data suggest that there may be a sex-specific effect which would be interesting to explore further, both in RPL and in a background population.

Maternal homozygocity for a 14 base pair insertion in exon 8 of the HLA-G gene and carriage of HLA class II alleles restricting HY immunity predispose to unexplained secondary recurrent miscarriage and low birth weight in children born to these patients.

Homozygous carriage of a 14 base pair (bp) insertion in exon 8 of the HLA-G gene may be associated with low levels of soluble HLA-G and recurrent miscarriage (RM). We investigated the G14bp insertion(ins)/deletion(del) polymorphism in 339 women with unexplained RM and 125 control women. In all patients and patients with secondary RM after a firstborn boy, 19.2% and 23.9%, respectively, were G14bp ins/ins compared with 11.2% of controls (p<0.05 and p<0.01). Among secondary RM patients with a firstborn boy, G14bp del/del and no carriage of an HLA class II (HYrHLA) allele restricting immunity against male-specific minor HY antigens was found less often than in controls (p<0.05) whereas G14bp ins/ins and carriage of HYrHLA predisposed (p<0.08) to this clinical entity. The mean birth weight of firstborn boys born to G14bp ins positive secondary RM patients was significantly lower than expected (p<0.001) but only in carriers of HYrHLA alleles (p<0.01). In conclusion, homozygosity for G14bp ins predisposes to RM. The combination of G14 ins homozygosity and carriage of HYrHLA predisposes to secondary RM in women with a firstborn boy and negatively affects birth weight in these boys.

Study of the structure and impact of human leukocyte antigen (HLA)-G-A, HLA-G-B, and HLA-G-DRB1 haplotypes in families with recurrent miscarriage.

A 14-base pair (bp) long insertion (ins)/deletion (del) polymorphism in exon 8 in the 3'-untranslated region of the human leukocyte antigen (HLA)-G gene is suggested to affect transcription of the gene. Carriage of the G14bp ins is associated with low levels of soluble HLA-G and increases the risk of recurrent miscarriage (RM). Due to existence of strong linkage disequilibrium (LD) in the HLA region, the primary susceptibility genes for RM in the HLA-G region have not yet been identified. HLA-A, -B, -DRB1, and -G14bp polymorphisms were investigated in 29 Caucasian families with two or more siblings suffering unexplained RM. Strong positive LD was detected between the G14bp ins and HLA-A*01, -A*11, -A*31, -B*08, and DRB1*03, whereas strong negative LD was found between G14bp ins and HLA-A*02, -A*03, and -A*24. The frequency of haplotypes with HLA-G14bp ins inherited from the mother was significantly increased in probands with RM (p = 0.05). The increased compatibility between probands and their mothers for maternal G14 ins positive haplotypes suggests that maternal-fetal compatibility for chromosomal segments adjacent to HLA-G locus is a risk factor for female offspring to experience RM in their later reproductive life.

IGF2 mRNA-binding protein 2: biological function and putative role in type 2 diabetes.

Recent genome-wide association (GWA) studies of type 2 diabetes (T2D) have implicated IGF2 mRNA-binding protein 2 (IMP2/IGF2BP2) as one of the several factors in the etiology of late onset diabetes. IMP2 belongs to a family of oncofetal mRNA-binding proteins implicated in RNA localization, stability, and translation that are essential for normal embryonic growth and development. This review provides a background to the IMP protein family with an emphasis on human IMP2, followed by a closer look at the GWA studies to evaluate the significance, if any, of the proposed correlation between IMP2 and T2D.

Future directions of failed implantation and recurrent miscarriage research.

Recurrent implantation failure is today the major reason for women completing several IVF/intracytoplasmic sperm injection attempts without having achieved a child, and is probably also the explanation for many cases of unexplained infertility. Most causes of recurrent miscarriage are still poorly elucidated, but from a theoretical point of view recurrent implantation failure and recurrent miscarriage are suggested to have partly overlapping causes. Recent research has indeed documented that both syndromes can be caused by the same embryonic chromosomal abnormalities and the same maternal endocrine, thrombophilic and immunological disturbances. Consequently, many treatments attempting to normalize these abnormalities have been tested or are currently used in women with both recurrent implantation failure and recurrent miscarriage. However, no treatment for the two syndromes is at the moment sufficiently documented to justify its routine use. In this review, an overview is given regarding present knowledge about causes that may be common for recurrent implantation failure and recurrent miscarriage, and suggestions are put forward for future research that may significantly improve understanding and treatment options for the syndromes.

Inflammation and miscarriage.

Most relevant studies in animals and humans indicate that some degree of systemic or uterine inflammation is necessary both for normal implantation and pregnancy. However, if inflammation becomes too excessive it might cause pregnancy complications such as fetal resorption/miscarriage. The main regulator of the correct level of inflammation at the feto-maternal interface seems to be the uterine CD16(-) CD56(bright) natural killer (NK) cells. Trophoblast debris, apoptotic cells and progesterone probably stimulate/regulate the production of inflammatory cytokines from these cells. Miscarriage of karyotypically normal embryos may occur when the level of inflammation at the feto-maternal interface falls outside the optimal range. This may be caused by an insufficient influx of CD56(bright) NK cells into the decidua, too little soluble histocompatibility leukocyte antigen (HLA)-G secretion from the trophoblast, hypersecretion of inflammatory cytokines due to the presence of high-production polymorphisms, presence of maternal HLA-DR alleles associated with high tumor necrosis factor (TNF)-alpha production, or maternal mannose-binding lectin deficiency.