Kristiina Rull

Genetics of Recurrent Miscarriage: Challenges, Current Knowledge, Future Directions

Recurrent miscarriage (RM) occurs in 1–3% of couples aiming at childbirth. Due to multifactorial etiology the clinical diagnosis of RM varies. The design of genetic/“omics” studies to identify genes and biological mechanisms involved in pathogenesis of RM has challenges as there are several options in defining the study subjects (female patient and/or couple with miscarriages, fetus/placenta) and controls. An ideal study would attempt a trio-design focusing on both partners as well as pregnancies of the couple. Application of genetic association studies focusing on pre-selected candidate genes with potential pathological effect in RM show limitations. Polymorphisms in ∼100 genes have been investigated and association with RM is often inconclusive or negative. Also, implication of prognostic molecular diagnostic tests in clinical practice exhibits uncertainties. Future directions in investigating biomolecular risk factors for RM rely on integrating alternative approaches (SNPs, copy number variations, gene/protein expression, epigenetic regulation) in studies of single genes as well as whole-genome analysis. This would be enhanced by collaborative network between research centers and RM clinics.

Genomics and genetics of gonadotropin beta-subunit genes: Unique FSHB and duplicated LHB/CGB loci.

The follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (HCG) play a critical role in human reproduction. Despite the common evolutionary ancestry and functional relatedness of the gonadotropin hormone beta (GtHB) genes, the single-copy FSHB (at 11p13) and the multi-copy LHB/CGB genes (at 19q13.32) exhibit locus-specific differences regarding their genomic context, evolution, genetic variation and expressional profile. FSHB represents a conservative vertebrate gene with a unique function and it is located in a structurally stable gene-poor region. In contrast, the primate-specific LHB/CGB gene cluster is located in a gene-rich genomic context and demonstrates an example of evolutionary young and unstable genomic region. The gene cluster is shaped by a constant balance between selection that acts on specific functions of the loci and frequent gene conversion events among duplicons. As the transcription of the GtHB genes is rate-limiting in the assembly of respective hormones, the genomic and genetic context of the FSHB and the LHB/CGB genes largely affects the profile of the hormone production.

The evolution and genomic landscape of CGB1 and CGB2 genes

The origin of completely novel proteins is a significant question in evolution. The luteinizing hormone (LHB)/chorionic gonadotropin (CGB) gene cluster in humans contains a candidate example of this process. Two genes in this cluster (CGB1 and CGB2) exhibit nucleotide sequence similarity with the other LHB/CGB genes, but as a result of frameshifting are predicted to encode a completely novel protein. Our analysis of these genes from humans and related primates indicates a recent origin in the lineage specific to humans and African great apes. While the function of these genes is not yet known, they are strongly conserved between human and chimpanzee and exhibit three-fold lower diversity than LHB across human populations with no mutations that would disrupt the coding sequence. The 5′-upstream region of CGB1/2 contains most of the promoter sequence of hCGβ plus a novel region proximal to the putative transcription start site. In silico prediction of putative transcription factor binding sites supports the hypothesis that CGB1 and CGB2 gene products are expressed in, and may contribute to, implantation and placental development.

Haplotype Structure of FSHB, the Beta-Subunit Gene for Fertility-Associated Follicle-Stimulating Hormone: Possible Influence of Balancing Selection

Follicle‐stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor‐binding beta‐subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76‐96.6% of each populations sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organisms fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.

Differential Expression Profile of Growth Hormone/Chorionic Somatomammotropin Genes in Placenta of Small- and Large-for-Gestational-Age Newborns

CONTEXT The human growth hormone/chorionic somatomammotropin (hGH/CSH) locus at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placenta-expressed genes (GH2, CSH1, CSH2, and CSHL1), is implicated in regulation of postnatal and intrauterine growth. A positive correlation has been reported between the offsprings birth weight and serum placental GH (coded by GH2) and placental lactogen (coded by CSH1, CSH2) levels in pregnant women. OBJECTIVE The objective of the study was the investigation of the hypothesis that the mRNA expression profile of placental hGH/CSH genes contributes to the determination of birth weight. DESIGN AND SUBJECTS We developed a sensitive, fluorescent-labeled semiquantitative RT-PCR assay coupled with gene-specific restriction analysis, capable of distinguishing alternative splice-products of individual placental hGH/CSH genes and quantification of their relative expression levels. The detailed profile of alternative transcripts of GH2, CSH1, CSH2, and CSHL1 genes in placenta from uncomplicated term pregnancies of the REPROMETA sample collection was addressed in association with the birth weight of newborns, grouped as appropriate for gestational age (AGA; n = 23), small for gestational age (SGA; n = 15), and large for gestational age (LGA; n = 34). RESULTS The majority of pregnancies with SGA newborn showed down-regulation of the entire hGH/CSH cluster in placenta, whereas in the case of LGA, the expression of CSH1-1, CSH2-1, and CSHL1-4 mRNA transcripts in placenta was significantly increased compared with AGA newborns (P < 0.0001, P = 0.009, P = 0.002, respectively). CONCLUSION The expression profile of placental hGH/CSH genes in placenta is altered in pregnancies accompanied by SGA and LGA compared with AGA newborns, and thus, it may directly affect the circulating fetal and maternal placental GH and placental lactogen levels.

IgG, IgA and IgM Antibodies against FSH: Serological Markers of Pathogenic Autoimmunity or of Normal Immunoregulation?

Autoimmune mechanisms are often involved in causing infertility. Among the possible targets of autoantibodies, the follicle‐stimulating hormone (FSH) which regulates the follicular maturation in human ovary is a promising candidate. We aimed to study whether anti‐FSH‐antibodies might be involved in different clinical types of infertility.

Chorionic Gonadotropin β-Gene Variants Are Associated with Recurrent Miscarriage in Two European Populations

CONTEXT The incidence of recurrent miscarriage (RM) (>or=3 consecutive pregnancy losses) is estimated as 1-2% in fertile couples. Familial clustering of RM has suggested the contribution of a genetic component. OBJECTIVE A low level of human chorionic gonadotropin (HCG) in maternal serum during the first trimester of the pregnancy is a clinically accepted risk factor for miscarriage. We sought to study whether variation in chorionic gonadotropin beta-subunit genes (CGBs) expressed in placenta may contribute to the risk of RM. DESIGN Resequencing of CGB5 and CGB8, the two most actively transcribed loci of the four HCG beta-duplicate genes, was performed. SETTING A case-control study involving two sample sets, from Estonia (n = 194) and Finland (n = 185), was performed. PATIENTS RM patients (n = 184) and fertile controls (n = 195) participated in the study. RESULTS From 71 identified variants in CGB5 and CGB8, 48 polymorphisms were novel. Significant protective effect was associated with two single nucleotide polymorphisms located at identical positions in intron 2 in both CGB5 [P = 0.007; odds ratio (OR) = 0.53] and CGB8 (P = 0.042; OR = 0.15), and with four CGB5 promoter variants (P < 0.03; OR = 0.54-0.58). The carriers of minor alleles had a reduced risk of RM. The haplotype structure of the CGB8 promoter was consistent with balancing selection; a rare mutation in CGB8 initiator element was detected only among patients (n = 3). In addition, three rare nonsynonymous substitutions were identified among RM cases as possible variants increasing the risk of recurrent pregnancy loss. CONCLUSION The findings encourage studying the functional effect of the identified variants on CGB expression and HCG hormone activity to elucidate further the role of CGB variation in RM.

Mid-Gestational Gene Expression Profile in Placenta and Link to Pregnancy Complications

Despite the importance of placenta in mediating rapid physiological changes in pregnancy, data on temporal dynamics of placental gene expression are limited. We completed the first transcriptome profiling of human placental gene expression dynamics (GeneChips, Affymetrix®; ∼47,000 transcripts) from early to mid-gestation (n = 10; gestational weeks 5–18) and report 154 genes with significant transcriptional changes (ANOVA, FDR P<0.1). TaqMan RT-qPCR analysis (n = 43; gestational weeks 5–41) confirmed a significant (ANOVA and t-test, FDR P<0.05) mid-gestational peak of placental gene expression for BMP5, CCNG2, CDH11, FST, GATM, GPR183, ITGBL1, PLAGL1, SLC16A10 and STC1, followed by sharp decrease in mRNA levels at term (t-test, FDR P<0.05). We hypothesized that normal course of late pregnancy may be affected when genes characteristic to mid-gestation placenta remain highly expressed until term, and analyzed their expression in term placentas from normal and complicated pregnancies [preeclampsia (PE), n = 12; gestational diabetes mellitus (GDM), n = 12; small- and large-for-gestational-age newborns (SGA, LGA), n = 12+12]. STC1 (stanniocalcin 1) exhibited increased mRNA levels in all studied complications, with the most significant effect in PE- and SGA-groups (t-test, FDR P<0.05). In post-partum maternal plasma, the highest STC1 hormone levels (ELISA, n = 129) were found in women who had developed PE and delivered a SGA newborn (median 731 vs 418 pg/ml in controls; ANCOVA, P = 0.00048). Significantly higher expression (t-test, FDR P<0.05) of CCNG2 and LYPD6 accompanied with enhanced immunostaining of the protein was detected in placental sections of PE and GDM cases (n = 15). Our study demonstrates the importance of temporal dynamics of placental transcriptional regulation across three trimesters of gestation. Interestingly, many genes with high expression in mid-gestation placenta have also been implicated in adult complex disease, promoting the discussion on the role of placenta in developmental programming. The discovery of elevated maternal plasma STC1 in pregnancy complications warrants further investigations of its potential as a biomarker.

Differential placental expression profile of human Growth Hormone/Chorionic Somatomammotropin genes in pregnancies with pre-eclampsia and gestational diabetes mellitus

Highlights ► A trend for lower level of all placental hGH/CSH transcripts in preeclampsia. ► Reduced GH2-2/CSH1-2 transcripts retaining intron 4 only in preeclampsia without SGA. ► Increased level of placental GH2 mRNA in gestational diabetes with LGA newborns. ► hGH/CSH genes exhibit pleiotropic effects on fetal growth and maternal metabolism.

Extensive shift in placental transcriptome profile in preeclampsia and placental origin of adverse pregnancy outcomes

One in five pregnant women suffer from gestational complications, prevalently driven by placental malfunction. Using RNASeq, we analyzed differential placental gene expression in cases of normal gestation, late-onset preeclampsia (LO-PE), gestational diabetes (GD) and pregnancies ending with the birth of small-for-gestational-age (SGA) or large-for-gestational-age (LGA) newborns (n = 8/group). In all groups, the highest expression was detected for small noncoding RNAs and genes specifically implicated in placental function and hormonal regulation. The transcriptome of LO-PE placentas was clearly distinct, showing statistically significant (after FDR) expressional disturbances for hundreds of genes. Taqman RT-qPCR validation of 45 genes in an extended sample (n = 24/group) provided concordant results. A limited number of transcription factors including LRF, SP1 and AP2 were identified as possible drivers of these changes. Notable differences were detected in differential expression signatures of LO-PE subtypes defined by the presence or absence of intrauterine growth restriction (IUGR). LO-PE with IUGR showed higher correlation with SGA and LO-PE without IUGR with LGA placentas. Whereas changes in placental transcriptome in SGA, LGA and GD cases were less prominent, the overall profiles of expressional disturbances overlapped among pregnancy complications providing support to shared placental responses. The dataset represent a rich catalogue for potential biomarkers and therapeutic targets.