Identification Of Copy Number Variants In Couples With Unexplained Recurrent Miscarriage And Predictive Value Of HCG In Pregnancy Outcome

Part Ⅰ. Identification of copy number variants in couples with unexplained recurrent miscarriageBackgrounds:Recurrent miscarriage (RM) is defined as three or more consecutive pregnancy losses. RM affects1%of couples. Unexplained recurrent miscarriage (URM) is defined as three or more consecutive pregnancy losses without identifiable factors which included abnormal chromosomes, uterine abnormalities, endocrine disorders, infection and immune factors. RM seriously affects women’s reproductive health and has a negative effect on both patients and their families on many psychological and physiological levels. The lack of pertinence in URM treatment presents a significant challenge to the treatment of clinical doctors and the reproductive health of URM patients.At present, the studies at the molecular level for URM are carried out to mostly evaluate the relationship between cytokines, angiogenic mediators, and hormones gene polymorphisms and URM. But the relevance of gene polymorphisms and the development of URM is still unclear. Cytokines, angiogenic mediators, and hormones are involved in all stages of reproduction and pregnancy outcome. Now impaired production and/or unbalanced ratios of these mediators are considered to be implicated in the pathogenesis of URM. There have been several studies aimed to investigate the relationship between cytokines, angiogenic mediators, and hormones gene polymorphisms and URM. But up to present time, the results of these studies are mostly contradictory, and few significant associations have been identified.The concept of copy number varients (CNVs) has been put forward in recent years. A CNV is now operationally defined as a DNA segment, longer than1kb, with a variable copy number compared with a reference genome. It has been shown that CNVs may participate in the pathogenesis of URM. CNVs inherited from a carrier parent may lead to RM if (i) the CNV contains imprinted genes;(ii) genes involved in the CNV are relevant for embryonic/placenta growth and have a mutation in the other allele; or (iii) genes in the CNV are variably expressed.Array-based comparative genomic hybridization (array-CGH) is a recently developed molecular karyotyping technology permiting the simultaneous rapid high resolution genome-wide CNVs analysis and mapping of DNA sequences by a hybridization experiment. So it has been admitted to be the most effective tool for detecting chromosome microdeletions and microduplications.With the advent and application of array-CGH, the results of CNVs analysis of many complex common diseases have been reported.There have been no a large scale of CNVs analysis of URM, due to the filtering stringency of cases, the limited number of cases and technical difficulties.Objective:To investigate CNVs in couples with URM.Methods:Array-CGH was used to assess for CNVs in peripheral blood samples of50URM couples and38couples with at least one live birth and no history of pregnancy pathologies.Results:The rates of CNVs from7q21.3, Xp22.31and Xp22.33in one or both URM couples were markedly higher than controls (22.0%vs2.6%;44.0%vs18.0%;90.0%vs71.0%, P<0.05). As significant CNVs regions of URM, the sizes of CNVs from7q21.3, Xp22.31, Xp22.33were no statistical differences between URM couples and controls. Conclusions:The study shows that the significant CNVs regions of URM are from 7q21.3, Xp22.31and Xp22.33. Further investigations of CNVs, particularly those involving candidate URM genes, in couples with URM could be worthwhile. Part Ⅱ. Serum HCG level measured17days after oocyte retrieval can predict final clinical pregnancy outcomes in IVF/ICSI treatment cyclesBackgrounds:In1978, the first infant through in vitro fertilization-embryo transfer (IVF-ET) was born. Since then, the use of assisted reproductive technology (ART) has been increased substantially. ART is regarded as the last treatment for infertile couples, and it has been shown that couples undergoing ART have an increased risk of adverse pregnancy outcomes in comparison with normal population. So couples undergoing ART are often plagued by stress and anxiety related to their treatment and subsequent pregnancy outcome.Thus, an early, reliable predictor of pregnancy outcome following ART,is of great importantce to both clinicians and infertile couples undergoing ART.Serum markers and transvaginal ultrasound examination are part of the routine follow-up after IVF-ET. But the gestational sac by transvaginal ultrasound examination is reliably visible after the third week after ET. Serum markers are frequently-used because it can confirm the establishment of pregnancy and forecast its outcome after ART at the earlier stage. A number of studies have investigated the relationship between serum markers and pregnancy outcome after IVF, such as human chorionic gonadotropin (HCG), progesterone, oestradiol and inhibin.HCG, a glycoprotein hormone, can be detected in the maternal serum as early as approximately6to8days after fertilization. Being produced and secreted by syncytial trophoblast, its serum level represents the trophoblastic mass and function. The levels of maternal HCG doubles approximately every2days in the first2months of a normal gestation. Falling serum HCG levels or levels that do not double within2days are used to indicate a failing pregnancy in the first trimester.A number of authors have suggested that maternal levels of HCG are useful to distinguish between normal and abnormal pregnancy outcome after ART, but there are still some limitations and imprecision, such as the limited number of ART cycles, the different standards of the time of initial serum HCG measurement. What’s more, many of the previous studies have analyzed the relationship between serum HCG levels and pregnancy outcomes (mainly about biochemical loss, ectopic pregnancy, spontaneous abortion and ongoing pregnancies) after ART, but few about final clinical pregnancy outcomes, i.e. live birth. These factors may limit the comparability of results in the previous studies and diminish the predictive value of early serum HCG levels.Objective:To investigate whether serum HCG levels measured17days after oocyte retrieval (OR) can predict final clinical pregnancy outcomes in IVF/intracytoplasmic sperm injection-embryo transfer (ICSI-ET) cycles.Methods:Serum HCG levels17days after OR and the subsequent clinical pregnancy outcomes in6560patients with positive serum HCG were analysed.Results:Of6560patients, patients with positive, but low serum HCG levels (<100IU/L) had an increased risk of abnormal clinical pregnancy outcomes (spontaneous miscarriage or ectopic pregnancy)(P<0.05). Compared with abnormal clinical pregnancy, normal clinical pregnancy (live birth) showed significantly higher HCG levels at17days after OR (596.80IU/L vs277.80IU/L, P<0.001). The HCG level of live birth was markedly higher than spontaneous miscarriage (596.80IU/L vs357.15IU/L, P<0.001) and ectopic pregnancy (596.80IU/L vs129.30IU/L, P<0.001), respectively. The cut-off value was377.8IU/L to predict live birth with0.730area under the receiver operating characteristic (ROC curve)(95%CI=0.713-0.747,75.9%sensitivity,61.2%specificity, P<0.001).Conclusions:Serum HCG levels measured on the17th day after OR are clinically useful in predicting final clinical pregnancy outcomes in IVF/ICSI-ET cycles. However, it is important to note that no HCG cut-off has a sensitivity or specificity of100%for either normal or abnormal pregnancies, which makes it essential to continue routine monitoring of ART pregnancy outcomes.