Classification of factors involved in nonreportable results of noninvasive prenatal testing (NIPT) and prediction of success rate of second NIPT.

OBJECTIVE: To evaluate the reasons for nonreportable cell-free DNA (cfDNA) results in noninvasive prenatal testing (NIPT), we retrospectively studied maternal characteristics and other details associated with the results. METHODS: A multicenter retrospective cohort study in pregnant women undergoing NIPT by massively parallel sequencing (MPS) with failed cfDNA tests was performed between April 2013 and March 2017. The women's data and MPS results were analyzed in terms of maternal characteristics, test performance, fetal fraction (FF), z scores, anticoagulation therapy, and other details of the nonreportable cases. RESULTS: Overall, 110 (0.32%) of 34 626 pregnant women had nonreportable cfDNA test results after an initial blood sampling; 22 (20.0%) cases had a low FF (<4%), and 18 (16.4%) cases including those with a maternal malignancy, were found to have altered genomic profile. Approximately half of the cases with nonreportable results had borderline z score. Among the women with nonreportable results because of altered genomic profile, the success rate of retesting using a second blood sampling was relatively low (25.0%-33.3%). Thirteen (11.8%) of the women with nonreportable results had required hypodermic heparin injection. CONCLUSIONS: The classification of nonreportable results using cfDNA analysis is important to provide women with precise information and to reduce anxiety during pregnancy.

Factors affecting parental decisions to terminate pregnancy in the presence of chromosome abnormalities: a Japanese multicenter study.

OBJECTIVE: To investigate the rates of termination of pregnancy (TOP) for fetal chromosomal abnormalities and factors related to such parental decision in Japan. METHODS: A multicenter retrospective cohort study of chromosomal abnormalities diagnosed before 22 weeks of gestation between April 2008 and March 2015. The pregnancy outcomes and parental decisions were investigated. RESULTS: Among 931 fetuses with chromosome abnormalities, the total TOP rate was 75.1% (699/931). TOP rates were 89.3% (585/655) in autosomal aneuploidies and 40.8% (51/125) in sex chromosome aneuploidies. Trisomy 21 showed the highest TOP rate (93.8% [390/416]) followed by trisomy 18 (84.5% [163/193]) and trisomy 13 (71.9% [23/32]). Indications for karyotyping were related to a parental decision for TOP (p < 0.01): in cases of autosomal aneuploidy, with fetal abnormal ultrasound findings as the reference value, diagnoses made following positive results at non-invasive prenatal testing (adjusted odds ratio [OR]: 13.7, 95% confidence interval [CI] 4.07-45.9) and those because of advanced maternal age (adj. OR 2.91, 95% CI 1.15-7.35) were significantly more frequent. CONCLUSIONS: In Japan, pregnancies with fetal trisomy 21 are more likely to result in TOP when diagnosed in utero than any other chromosome anomaly. The indications for prenatal karyotyping strongly affect the decision to TOP. © 2016 John Wiley & Sons, Ltd.

Mutational analysis of FOXL2 p.C134W and expression of bone morphogenetic protein 2 in Japanese patients with granulosa cell tumor of ovary.

AIM: To assess whether FOXL2 p.C134W mutation may play a role in the development of human ovarian tumors in the Japanese, we investigated the FOXL2 codon 134 mutation and protein expression of inhibin-α, bone morphogenetic protein 2 (BMP2) and follistatin (FST) in Japanese patients with granulosa cell tumor (GCT) of the ovary and other ovarian tumors. METHODS: We analyzed 114 tumor tissues from ovarian tumors, including 44 adult-type and two juvenile-type GCT of the ovary and 68 ovarian tumors by DNA sequencing. Immunohistochemistry was also performed in the adult and juvenile GCT tissues by immunostaining inhibin-α, BMP2 and FST. RESULTS: We found the FOXL2 p.C134W mutation in 27 out of 44 (61.4%) adult-type GCT of the ovary, but none in other ovarian tumors. Histologically, all of the adult-type GCT sections were positive for inhibin-α, and the expression of BMP2 and FST was detected in 14 of 44 (31.8%) and zero of 47 (0%), respectively. No significant differences regarding the diagnosed age, preoperative serum carbohydrate antigen 125 levels, or BMP2 immunopositivity between the FOXL2 p.C134W mutation-positive and mutation-negative were found in the adult-type GCT patients. CONCLUSION: Our findings suggest that FOXL2 p.C134W mutation-positive adult-type GCT of the ovary may not be common in the Japanese as compared to the previous data.

Role of cathepsin E in decidual macrophage of patients with recurrent miscarriage.

In a previous study, we reported that the cathepsin-cystatin system caused endometrial dysfunction in early pregnancy. Here, we investigated the existence and contribution of cathepsin E in early pregnancy in patients with recurrent miscarriage (RM). The effect of cathepsin deficiency on fertility and female reproductive organs were also analyzed in CatE(-/-) mice. Human studies were conducted in a hospital setting, with informed consent. Cervical mucus was collected from RM patients in early pregnancy (4-6 gestational weeks, n = 21), and the pregnancy outcome was compared prospectively. The cathepsin E expression in decidua of RM patients (n = 49) and normal pregnant women undergoing elective surgical abortion (n = 24) was measured using SDS-PAGE, and western blot analysis. Decidual macrophages were isolated from RM patients (n = 6) and stimulated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Results from the mouse model showed that CatE(-/-) mice were fertile, but the litter number was significantly smaller. The uterus of CatE(-/-) mice showed granulation tissue. In human samples, protease activity of cathepsin E measured with Fluorescence-Quenching Substrate (KYS-1) in cervical mucus of patients who developed miscarriage was markedly decreased compared with patients without RM. The expression of cathepsin E in decidua, semi-quantified by SDS-PAGE, western blot analysis was significantly lower in RM patients compared with patients without RM. By double staining immunofluorescence, the staining of cathepsin E was observed in CD14 or CD68 positive cells in all deciduas. Upon stimulation with LPS and IFN-γ, the expression of cathepsin E in cell lysate of decidual macrophages was markedly reduced in RM patients compared with controls. The results suggested that decreased activity of cathepsin E produced by decidual macrophages might be responsible for the induction of miscarriages in some RM patients.

Prenatal diagnosis of X-linked recessive Lenz microphthalmia syndrome.

Lenz microphthalmia syndrome comprises microphthalmia-anophthalmia with mental retardation, malformed ears and skeletal anomalies, and is inherited in an X-linked recessive pattern. In 2004, it was reported that the missense mutation (BCL-6 co-repressor gene [BCOR] c.254C>T, p.P85L) in a single family with Lenz microphthalmia syndrome co-segregated with the disease phenotype. We report a case of prenatal diagnosis for X-linked recessive Lenz microphthalmia syndrome with the mutation. A 32-year-old gravida 5, para 2 Japanese woman was referred to Nagoya City University Hospital at 15 weeks of gestation. After genetic counseling and informed consent, amniocentesis was performed for fetal karyotyping, which was 46,XY. Using the extracted DNA from cultured amniotic cells, fetal search for BCOR c.254C>T mutation was undertaken. The couple requested medical termination of pregnancy, and the postabortion examination confirmed the diagnosis. This is the third report of a BCOR mutation, associated with X-linked syndromic microphthalmia, and most importantly, it is always the same mutation. The prenatal genetic diagnosis of the Lenz microphthalmia syndrome allowed time for parental counseling and delivery planning.

Müllerian anomalies and recurrent miscarriage.

PURPOSE OF REVIEW: To review the prevalence of congenital uterine anomalies and pregnancy outcomes in patients with these anomalies. RECENT FINDINGS: Women with a history of recurrent miscarriage have been estimated to have a 3.2-10.4% likelihood of having a major uterine anomaly except arcuate uterus. Hysterosalpingography and/or 2D ultrasound can be used as the initial screening tools. The American Fertility Society classification of Müllerian anomalies is the most commonly utilized standardized classification. However, there is still no international consensus to distinguish between septate and bicornuate uteri. A total of 35.1-65.9% of patients with bicornuate or septate uteri give live births after correctional surgery. In regard to the live birth rate in the absence of surgery, it has been reported that 33.3-59.5% of patients with such anomalies had a successful first pregnancy after the examination, as compared to 71.7% of individuals with normal uteri (P=0.084), with no significant difference in the cumulative live birth rate (78.0 and 85.5%, respectively) between the two groups. SUMMARY: Randomized controlled trials comparing the pregnancy outcomes between cases treated and not treated by surgery among patients with a history of recurrent miscarriage are needed because it is not established whether surgery could improve live birth rate.

Two cases of prenatally diagnosed sacrococcygeal teratoma type I with different clinical features.

Sacrococcygeal teratoma (SCT) is a rare congenital disease and prognostic factors have not been entirely established. We report two cases of fetal SCT with different clinical courses. Case 1 was a cystic, slow growing tumor with mild vascularity. The tumor was removed one week after delivery at 35 weeks, and there was no recurrence at 1.5-year follow-up. Case 2 was a solid, rapid growing tumor with rich vascularity. Cesarean section was performed due to severe fetal hydrops and mirror syndrome in the mother at 27 weeks. The tumor had ruptured and was removed soon after delivery to control bleeding, but the baby died the next day. Our cases suggest that solid component and rich vascularity might correlate with poor prognosis.

Uterine anomaly and recurrent pregnancy loss.

Women with recurrent pregnancy loss have a 3.2 to 6.9% likelihood of having a major uterine anomaly and a 1.0 to 16.9% chance of having an arcuate uterus. Bicornuate and septate uterine have a negative impact on reproductive outcomes and are associated with subsequent euploid miscarriage. The impact of an arcuate uterus on pregnancy outcome remains unclear. There are no definitive criteria to distinguish among the arcuate, septate, and bicornuate uteri. The American Fertility Society classification of Müllerian anomalies is the most common standardized classification of uterine anomalies. According to estimates, 65 to 85% of patients with bicornuate or septate uteri have a successful pregnancy outcome after metroplasty. However, 59.5% of the patients with such anomalies have a successful subsequent pregnancy without surgery, with a cumulative live birthrate of 78.0%. There is no case-control study to compare live birthrates in women who had surgery compared with those who did not. Strict criteria to distinguish between the bicornuate and septate uterus should be established. Further study is needed to confirm the benefits of metroplasty.

Prenatal diagnosis of familial lethal hypophosphatasia using imaging, blood enzyme levels, chorionic villus sampling and archived fetal tissue.

Hypophosphatasia is an inheritable disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia. A 33-year-old, gravida 4, para 3 Japanese woman was referred to Nagoya City University Hospital for prenatal genetic counseling because of two previous occurrences of fetal bone anomalies. The molecular examination showed that the fetus was homozygous for the TNSALP gene mutation c.1559delT, each parent being heterozygous. Genetic counseling was offered and at the next pregnancy, chorionic villus sampling was performed, whereupon genetic analysis confirmed that the fetus did not carry the familial mutation c.1559delT. Postnatal molecular genetic analysis using the cord tissue can provide a diagnosis of lethal hypophosphatasia and prenatal genetic diagnosis of the TNSALP gene allows time for parental counseling and delivery planning.

Glycogen storage disease type Ia (GSD Ia) during pregnancy: report of a case complicated by fetal growth restriction and preeclampsia.

Glycogen storage disease type Ia (GSD Ia) leads to disturbed glycogenolysis and gluconeogenesis due to a deficiency in the enzyme glucose-6-phosphatase. A patient with GSD Ia showed hypoglycemia and proteinuria without dietary management since early pregnancy. The patient's condition was complicated by hypertension with increase in proteinuria at 22 weeks of gestation. In spite of administration of antihypertensive drugs and dietary management, the disease became more severe with deterioration in the fetal status and inhibition of fetal growth. Thus, a cesarean section was performed at 26 weeks of gestation. The delivered male infant weighing 412 g died at 2 days after birth. The patient's blood pressure had normalized within 3 months after delivery, while proteinuria persisted.

The biological investigation of prostacyclin in preeclamptic women seen reduced endothelial function.

OBJECTIVE: Our aim was to determine the biological investigation of prostacyclin in preeclamptic women seen reduced endothelial vasodilatation by non-invasive technique in vivo. METHODS: Using a high resolution ultrasound transducer, diameters of brachial arteries were determined after reactive hyperemia in 15 non-pregnant, 20 normotensive pregnant and 20 preeclamptic women. The concentrations of 6-keto-prostaglandin F 1alpha (6keto-PGF 1alpha) in plasma and the concentrations of adenosine-3', 5'-cyclic monophosphate (cyclic AMP) in platelets and serum were measured among the groups. RESULTS: Flow-mediated vasodilatation at 1 min after reactive hyperemia was higher in normotensive pregnant than in the non-pregnant or preeclamptic women. The plasma concentration of 6 keto-PGF 1alpha as well as the serum concentration of cyclic AMP were lower in preeclamptic than those in normotensive pregnant women. The increase in cyclic AMP in the presence of a prostacyclin analogue in platelets was seen at similar levels in all three groups. CONCLUSION: From these results, the concentrations of prostacyclin in plasma and cyclic AMP in serum might be low possibly due to reduced production of prostacyclin in preeclamptic women seen reduced endothelial function.

Prenatal diagnosis of persistent cloaca.

We report four cases of persistent cloaca diagnosed at 32-33 weeks of gestation. In cases of persistent cloaca, serial prenatal ultrasonography shows transient fetal ascites, enlarged cystic structures arising from the fetal pelvis. Our four cases of persistent cloaca were diagnosed prenatally. Persistent cloaca should be considered in any female fetus presenting with hydronephrosis and a large cystic lesion arising from the pelvis as assessed by ultrasound and magnetic resonance imaging. Neither pulmonary hypoplasia nor severe oligohydramnios were found in any of our four cases, and they each had a good prognosis. Prenatal diagnosis allows time for parental counseling and delivery planning at a tertiary care center for neonatal intensive care and pediatric surgery.

The polycystic ovary syndrome does not predict further miscarriage in Japanese couples experiencing recurrent miscarriages.

PROBLEM: It has been a matter of controversy whether the polycystic ovary syndrome (PCOS) is actually a causal factor of miscarriages because of the absence of internationally established criteria. We, therefore, in this study investigated whether PCOS and a polycystic ovary (PCO) morphology have predictive value for subsequent miscarriages using new International and Japanese criteria. METHOD OF STUDY: A total of 195 patients with a history of two consecutive first trimester miscarriages and without abnormal chromosomes in either partner, antiphospholipid antibodies or uterine anomalies, were examined. The prospective pregnancy outcome was compared between patients with and without PCOS, PCO morphology, elevated luteinizing hormone (LH), hyperandrogenism and obesity. RESULTS: Of a total of 195 patients, 56 (28.7%) miscarried subsequently. Three (1.5%) and 12 (6.2%) were diagnosed as suffering from PCOS by Japanese and International criteria respectively. There was no relation between a diagnosis of PCOS, PCO morphology, elevated LH, free testosterone or obesity and the subsequent miscarriage rate. CONCLUSION: A routine test for diagnosis of PCOS is not necessary in patients experiencing recurrent miscarriages because none of the related parameters examined in this study predicted subsequent miscarriage.

Preoperative serum thrombopoietin levels are higher in patients with ovarian cancer than with benign cysts.

OBJECTIVE: To assess the diagnostic relevance of serum thrombopoietin (TPO) levels, we compared serum TPO levels between the patients with ovarian cancer and benign ovarian cysts. We also correlated serum TPO concentrations in ovarian cancer patients to know prognostic factors such as disease stage, tumor grade, histological subtype, and residual tumor mass. STUDY DESIGN: Preoperative serum TPO levels were measured in women with epithelial ovarian cancer (n=51) and women with benign ovarian cysts (n=25) using an enzyme-linked immunosorbent assay. RESULTS: The serum TPO concentration was significantly elevated in the ovarian cancer patients as compared to the benign patient controls. Platelet counts and TPO in the serum did not correlate in the ovarian cancer patients. However, using a TPO cutoff of 90 pg/ml and a CA125 cutoff of 30 units/ml, when both markers were elevated, the specificity was as high as 92%. CONCLUSION: Serum TPO concentrations may have a role in the diagnostic clinical setting for discerning benign from malignant ovarian tumors.

Elevated serum RANTES levels in patients with ovarian cancer correlate with the extent of the disorder.

OBJECTIVE: To assess the clinical relevance of serum regulated upon activation, normal T-cell expressed and secreted (RANTES) levels in distinguishing patients with ovarian cancers from those with benign ovarian cysts, we measured its concentration with reference to the disease stage, pathological grading, histological subtype, and the residual tumor mass. METHODS: Preoperative serum RANTES levels were measured in women with invasive epithelial ovarian cancer (n = 52), borderline ovarian tumor (n = 6), benign ovarian cysts (n = 28), or normal controls (n = 12) using an enzyme-linked immunosorbent assay. RESULTS: The serum RANTES concentration was significantly elevated in the ovarian cancer patients (median 53 ng/ml, interquartile range 23-104 ng/ml) compared to the benign ovarian cyst patients as controls (38 ng/ml, 5-72 ng/ml) values correlating with the stage of disease and the extent of residual tumor mass. No significant correlation between CA125 and RANTES in the serum was observed in either the controls or the ovarian cancer patients. Using a RANTES cutoff of 45 ng/ml and a CA125 cutoff of 35 units/ml, when either marker was elevated, the specificity improved 94%. CONCLUSION: Our study suggest that preoperative serum RANTES levels may be useful in differentiating benign ovarian tumors from malignancy correlating with the extent of the disorder.

Increased granulocyte chemotactic protein-2 concentrations in peritoneal fluid of women with endometriosis.

BACKGROUND: To evaluate the release of granulocyte chemotactic protein-2 (GCP-2) into peritoneal fluid in women with endometriosis, we measured its concentration with reference to the disease stage and the phase of the menstrual cycle. METHODS: Surgery was scheduled in the proliferative or secretory phase of the menstrual cycle for 64 women with endometriosis (n = 38) or cystadenomas (n = 26). GCP-2 concentrations in the peritoneal fluid were measured using an enzyme-linked immunosorbent assay. RESULT: Our findings indicated elevated concentrations of GCP-2 in peritoneal fluid from women with endometriosis during the proliferative phase, which were positively correlated with the stage of endometriosis. CONCLUSION: Inflammation associated with endometriosis may be involved in the pathogenesis of the disease through increasing levels of peritoneal fluid GCP-2.

Use of serum secretory leukocyte protease inhibitor levels in patients to improve specificity of ovarian cancer diagnosis.

OBJECTIVE: To assess the clinical relevance of serum secretory leukocyte protease inhibitor (SLPI) levels in distinguishing patients with ovarian cancers from those with benign ovarian cysts, we determined concentrations with reference to the FIGO stage and other clinical characteristics. METHODS: Preoperative serum SLPI levels were measured in women with invasive epithelial ovarian cancer (n = 55), benign ovarian cysts (n = 25), or normal controls (n = 38) using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum SLPI concentration was significantly elevated in the ovarian cancer patients (median 67 ng/ml, interquartile range 26-124 ng/ml) as compared to the benign cyst patients (37 and 25-66 ng/ml) or healthy women (32 and 25-43 ng/ml). Using an SLPI cutoff of 50 ng/ml and a CA125 cutoff of 30 units/ml, with both markers elevated the sensitivity was 95%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 89% between the malignant and benign cyst patients. CONCLUSION: Serum SLPI levels could be useful for differentiating benign ovarian cysts from malignancies and to improve the specificity of diagnosis.

Elevated angiogenin levels in the peritoneal fluid of women with endometriosis correlate with the extent of the disorder.

OBJECTIVE: To assess the release of angiogenin into peritoneal fluid in women with and without endometriosis by measuring its concentration with reference to disease stage, presence of red lesions, and phase of the menstrual cycle. DESIGN: Retrospective study. SETTING: Nagoya City University Hospital. PATIENT(S): Sixty-four women with endometriosis (n = 38) and cystadenomas (n = 26) for whom surgery was scheduled in the proliferative or secretory phase of the menstrual cycle. INTERVENTION(S): Peritoneal fluid samples were obtained at laparotomy or laparoscopy. MAIN OUTCOME MEASURE(S): Angiogenin concentrations in the peritoneal fluid, as measured by ELISA. RESULT(S): Angiogenin concentration in the peritoneal fluid was markedly elevated in the endometriosis patients (median 515 ng/mL, interquartile range 151-1763 ng/mL) compared with the cystadenoma (control) patients (195 ng/mL, 98-324 ng/mL), with values correlating with the extent of the disease. No significant differences between the proliferative phase and the secretory phase were observed in either the controls or the endometriosis patients. CONCLUSION(S): The inflammation associated with endometriosis, through increasing levels of peritoneal fluid angiogenin, might promote angiogenesis for progression of the disease and correlate with the extent of the disorder.