Endothelin-1 production via placental (pro)renin receptor in a mouse model of preeclampsia.

INTRODUCTION: Preeclampsia (PE) pathogenesis is explained by the two-stage disorder theory. However, mechanisms underlying hypertension and proteinuria in PE remain unclear. The role of (pro)renin receptor (PRR) in PE pathology has received special attention. We examined endothelin-1 (ET-1) production via placental PRR in a PE mouse model. METHODS: At 14.5 day-post-coitum (DPC), we performed a reduced uterine perfusion pressure (RUPP) operation, ligating the uterine arteriovenous vessels in female mice. We also infused these mice with a PRR inhibitor, decoy peptide in the handle region of prorenin (HRP) for mice (NH2-RIPLKKMPSV-COOH). At 18.5 DPC, blood, urine, and placenta were collected; fetus and placenta were weighed. We evaluated placental hypoxia using quantitative polymerase chain reaction (PCR), with hypoxia-inducible factor-1α (HIF-1α) as index. We also evaluated PRR, transforming growth factor-ß1 (TGF-ß1), and ET-1 expression in the placenta using quantitative PCR and western blotting. ET-1 concentration in blood plasma was assessed using enzyme-linked immunosorbent assay. RESULTS: Blood pressure and proteinuria significantly increased, and fetal and placental weights decreased in RUPP mice. HIF-1α, PRR, TGF-ß1, and ET-1 expressions considerably increased in RUPP mice placentas. ET-1 concentration in RUPP mice blood plasma was markedly increased. PRR inhibitor suppressed these changes. DISCUSSION: In PE model mice that underwent RUPP treatment, placental hypoxia increased PRR and ET-1 expression suggesting a causative relationship between ET-1 and intracellular PRR signaling. RUPP treatment, when combined with HRP, reversed the effect of elevated ET-1 levels in the model. This study may help to elucidate the pathogenesis of PE considering PRR and ET-1.

Short stature in small-for-gestational-age offspring born to mothers with hypertensive disorders of pregnancy.

OBJECTIVE: To investigate the incidence and risk factors of small-for-gestational age (SGA) short stature at 2 and 3 years of age in SGA offspring born to women with hypertensive disorders of pregnancy (HDP). METHODS: We examined 226 women with HDP whose respective SGA offspring were delivered. RESULTS: Eighty offspring (41.2%) were diagnosed with SGA short stature. The prematurity before 32 weeks of gestation was the most significant factor for catch-up growth failure. CONCLUSION: In SGA offspring born to women with HDP, SGA short stature incidence was high, and the risk factor was prematurity before 32 weeks of gestation.

Postpartum pyomyoma due to Mycoplasma hominis: A case report.

Pyomyoma is a rare condition that causes fever and abdominal pain associated with pregnancy, especially in the postpartum period. An appropriate diagnosis and early medical intervention are required to prevent serious complications. A 38-year-old primigravida with uterine fibroids had fever from the 11th day after cesarean section. The fever did not resolve despite repeated daily administration of broad-spectrum ß-lactam antibiotics for 2 weeks. Although the physical examination did not show any lower abdominal pain, a pelvic magnetic resonance scan revealed degenerative fibroids, and myomectomy was performed. Yellow-greenish odorless pus inside the uterus was detected, and Mycoplasma hominis was detected in the pus culture. Mycoplasma species are resistant to broad-spectrum penicillin antibiotics and can cause pyomyoma. Pyomyomas may not cause uterine tenderness, and the causative organism may be difficult to identify; therefore, additional imaging studies should be considered.

Diffuse myometrium thinning and placenta accreta spectrum in a patient with systemic lupus erythematosus (SLE): a case report and review of the literature.

BACKGROUND: Cases of uterine wall thinning and placental abnormalities complicated with systemic lupus erythematosus (SLE) during pregnancy have been reported in Asian countries for ten years. Long-term steroid use can cause muscle degeneration, but the mechanism of myometrium thinning was not known. Through the review of published articles, this report is the first review of cases to discuss the pathogenesis and clinical features of thinned myometrium and placenta accreta spectrum (PAS) in pregnant patients with SLE. CASE PRESENTATION: A twenty-nine-year-old primigravida with a history of lupus enteritis and paralytic ileus had a natural conception after less than two years of steroid treatment. An ultrasonographic study showed a thin uterine wall with a widespread thick placenta on the entire surface of the uterine cavity in the third trimester. At the 39th gestational week, she underwent a cesarean section due to the failure of the uterus to contract, even though the injection of oxytocin. There were several engorged vessels on the surface of the anterior uterine wall at the time of laparotomy. We decided to perform a hysterectomy because diffuse PAS replaced her uterus. CONCLUSION: A review of reported cases and our case shows an unusual complication of SLE that might be related to the particular condition of the estrogen-mediated immune system. Clinicians should always pay attention to the possibility of uterine wall thinning as uterine atony and the structural abnormality of the placenta for SLE patients with the unscarred uterus.

Obesity's Influence on Insulin Resistance in Pregnant Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine metabolic disorder that is associated with high insulin resistance and obesity. However, ~70% of women with PCOS in Japan are non-obese. We retrospectively analyzed the cases of 163 Japanese women with PCOS who visited our Ob/Gyn department in 2006-2018 to determine which has a greater effect on insulin resistance: PCOS or obesity. We reviewed the women's medical records and calculated their insulin resistance and insulin secretion. The women's mean age and pre-pregnancy body mass index (BMI) were 30±5.8 years and 24.8±5.6 kg/m2, respectively; their mean ± SD fasting plasma glucose, 94.1±13.7 mg/dL; HOMA-IR, 2.1±2.0; QUICKI, 0.4±0.0; and HOMA-ß, 108.9±88.0%. Sixtyeight women were pregnant, and 37% (n=25) were obese (BMI ≥ 25 kg/m2). Obesity had a greater effect on insulin resistance: fasting plasma glucose F(1, 53)=6.134, p<0.05; fasting insulin F(1, 53)=31.606, p<0.01; HOMA-IR F(1, 53)=31.670, p<0.01; QUICKI F(1, 53)=16.156, p<0.01. There was no significant difference in values other than QUICKI and testosterone between the women with and without PCOS. Obesity thus had a greater effect on increased insulin resistance in pregnant women with PCOS. Further studies of the insulin resistance of non-obese women with PCOS is needed, as non-obese women with PCOS are common in Asia.

Prevalence of common aneuploidy in twin pregnancies.

The incidence of chromosomal abnormalities in twin pregnancies is not well-studied. In this retrospective study, we investigated the frequency of chromosomal abnormalities in twin pregnancies and compared the incidence of chromosomal abnormalities in dichorionic diamniotic (DD) and monochorionic diamniotic (MD) twins. We used data from 57 clinical facilities across Japan. Twin pregnancies of more than 12 weeks of gestation managed between January 2016 and December 2018 were included in the study. A total of 2899 and 1908 cases of DD and MD twins, respectively, were reported, and the incidence of chromosomal abnormalities in one or both fetuses was 0.9% (25/2899) and 0.2% (4/1908) in each group (p = 0.004). In this study, the most common chromosomal abnormality was trisomy 21 (51.7% [15/29]), followed by trisomy 18 (13.8% [4/29]) and trisomy 13 (6.9% [2/29]). The incidence of trisomy 21 in MD twins was lower than that in DD twins (0.05% vs. 0.5%, p = 0.007). Trisomy 21 was less common in MD twins, even when compared with the expected incidence in singletons (0.05% vs. 0.3%, RR 0.15 [95% CI 0.04-0.68]). The risk of chromosomal abnormality decreases in twin pregnancies, especially in MD twins.

A case report of successful vaginal delivery in a patient with severe uterine prolapse and a review of the healing process of a cervical incision.

BACKGROUND: The incidence of severe uterine prolapse during childbirth is approximately 0.01%. Moreover, to the best of our knowledge, no reports detail the healing process of the cervix during uterine involution. This report describes successful vaginal delivery and the healing process of postpartum uterine prolapse and cervical tears in a patient with severe uterine prolapse. CASE PRESENTATION: A patient in her 40s (gravida 3, para 1, abortus 1) with severe uterine prolapse successfully delivered a live female baby weighing 3190 g at 38 + 5 weeks of gestation by assisted vaginal delivery. Uterine prolapse had improved to approximately 2° by 2 months postoperatively. On postpartum day 4, during the healing process of cervical laceration, the thread loosened in a single layer of continuous sutures due to uterine involution, and poor wound healing was observed. The wound was subsequently re-sutured with a two-layer single ligation suture (Gambee suture + vertical mattress suture). However, on postpartum day 11, a large thread ball was hindering the healing of the muscle layer, which improved with re-suturing. CONCLUSION: Although vaginal delivery in a patient with severe uterine prolapse is possible in some cases, the cervix should be sutured, while considering cervical involution after delivery.

EG-VEGF Induces Invasion of a Human Trophoblast Cell Line via PROKR2.

Extravillous trophoblast (EVT) invasion is important for embryo implantation, placental development, and successful remodeling of the uterine spiral artery. Endocrine gland derived-vascular endothelial growth factor (EG-VEGF) and matrix metalloproteinases (MMPs) are implicated in EVT invasion; however, the high con-centrations found in pregnancy pathologies have not been investigated in non-tumor trophoblasts. The roles of EG-VEGF, prokineticin receptors (PROKR1/2), MMP-2, and MMP-9 in EVT invasion during spiral artery remodeling were evaluated using human EVT from HTR-8/SVneo cell lines. The expression of MMP-2, MMP-9, and mitogen-activated protein kinase (MAPK), and Akt pathways in HTR-8/SVneo cells treated with recom-binant EG-VEGF alongside anti-PROKR1 and/or anti-PROKR2 antibodies was evaluated using quantitative reverse transcription-PCR and western blotting. Wound-healing and cell invasion assays were performed to assess the migration and invasion of these treated cells. Interestingly, 20 nM EG-VEGF activated ERK1/2 sig-naling and upregulated MMP-2 and MMP-9. This effect was suppressed by anti-PROKR2 antibody via ERK1/2 downregulation. Anti-PROKR2 antibody inhibited the migration and invasion of EG-VEGF-stimulated HTR-8/SVneo cells. Elevated concentrations of EG-VEGF enhance EVT invasion in a human trophoblast cell line by upregulating MMP-2 and MMP-9 via PROKR2. These new insights into the regulation of epithelial cell invasion may help in developing therapeutic interventions for placental-related diseases during pregnancy.

Non-Invasive Prenatal Genetic Testing (NIPT) Leading to Prenatal Diagnosis of Trisomy 21 Mosaicism and 18q Deletion Syndrome: Two Cases.

NIPT is non-definitive testing to estimate the possibility that fetuses have trisomy 21, trisomy 18, or trisomy 13. However, in NIPT-positive and indeterminate cases, rare chromosomal disease may become apparent, requiring advanced genetic considerations and counseling skills. We experienced two such cases, a trisomy 21 mosaicism case triggered by NIPT-positive status and 18q deletion syndrome triggered by NIPT-indeterminate status. These cases have two clinical implications for NIPT. First, it was revealed that trisomy mosaicism might be found in NIPT-positive cases that have lower Z-Scores than those inferred from the fraction of fetal cfDNA in the case of standard trisomy. Second, it is possible that microdeletion syndrome could be the reason for an indeterminate NIPT result. Today's genetic counseling requires more expertise in ethics and communication as well as genetic science because NIPT can lead to totally unexpected results.

Clinical Course of 60 Cesarean Scar Pregnancies.

Early diagnosis and therapy are important in a cesarean scar pregnancy (CSP), which can cause uterine rupture with resultant massive bleeding. However, there are some reports of CSPs continued to term. The optimal management of CSPs remains unclear; therefore, we investigated the clinical courses of CSPs diagnosed and treated at perinatal institutions in the Chugoku and Shikoku regions of Japan. We enrolled 60 women diag-nosed with CSP at 21 institutions from January 2006 to December 2015. Of the 60 women diagnosed with CSP, 57 were treated. Pregnancy was terminated in 48 women and continued in 9. Thirteen women underwent transabdominal hysterectomy; they experienced no postoperative complications or allogeneic blood transfu-sion. Nine women received therapies such as dilation and curettage, and 26 received non-surgical therapies such as methotrexate and topical administration of potassium chloride. Among 9 women who chose to con-tinue with their CSP, 7 successfully delivered newborns, 2 had uterine ruptures in the second trimester, and all women required transabdominal hysterectomy. Diagnosis and therapy in the first trimester of pregnancy are important in the management strategy of a CSP. When continuing a CSP, the risk of uterine rupture and trans-abdominal hysterectomy must be considered.

hCG values and gestational sac size as indicators of successful systemic methotrexate treatment in cesarean scar pregnancy.

OBJECTIVE: To retrospectively investigate cesarean scar pregnancy (CSP) patients who received systemic methotrexate (MTX) and to clarify the criteria for administering systemic MTX to CSP patients. MATERIALS AND METHODS: Fifteen CSP patients who were initially treated with systemic MTX (50 mg/m2/week) were included. Nine patients, who needed a uterine artery embolization (UAE) or a laparotomy, including a transabdominal hysterectomy (TAH), were defined as the unsuccessful MTX group. Six patients who did not require UAE or a laparotomy were defined as the successful MTX group. Furthermore, the hCG cut-off value and the GS cut-off size at the time of CSP diagnosis, which differentiated successful and unsuccessful patients, were defined. MTX success rates were investigated by combining the hCG and gestational sac (GS) size cut-off values. RESULTS: The hCG cut-off value was 17757.0 mIU/mL, and the GS cut-off size was 10.4 mm. In patients with hCG values less than 17757.0 mIU/mL, the MTX success rate was 75.0%. Fewer patients needed UAE or a laparotomy compared to patients with hCG values higher than 17757.0 mIU/mL (P = 0.007). In patients with a GS size less than 10.4 mm, the MTX success rate was 80.0%. Fewer patients among them needed UAE or a laparotomy compared to those among patients with a GS size greater than 10.4 mm (P = 0.089). In patients with hCG values and GS sizes lower than the cut-off values, the MTX success rate was 80.0%. Fewer patients among them needed UAE or a laparotomy compared to those among patients with hCG values and/or GS sizes higher than the cut-off values, respectively (P = 0.010). CONCLUSION: Patients with hCG values less than 17757.0 mIU/mL and GS sizes less than 10.4 mm may have a greater chance of successful systemic MTX treatment when it is used as the first line of treatment for CSP.

The Spiral Trial: A multicenter, randomized, controlled trial of Spiral thread sutures versus conventional thread sutures to prevent thinning of uterine scars following elective cesarean section.

BACKGROUND: The aim of this randomized controlled trial (RCT) is to investigate whether Spiral-thread sutures are superior to conventional sutures (0-Vicryl) for preventing uterine scar thinning following elective cesarean section. METHODS: This multicenter, parallel-group RCT will be conducted in four hospitals across three medical regions in Japan to assess 200 women (≥20 years old) with singleton pregnancies who are scheduled to undergo cesarean sections. Eligible women will be randomly assigned (1:1 ratio) to receive either the conventional uterine suture continuous absorption thread, which is most commonly used in Japan, or the Spiral thread. The primary endpoint is the degree of scar thinning, measured by transvaginal ultrasonography 6-7 months postoperatively, to evaluate the position of the uterus (anterior or posterior tilt) and myometrial wound thickness. The degree of thinning will be compared between the groups, and four measurements (mm) of the thinning area, including caudal distance, depth of the depression, remaining thickness of the myometrium on the serous side of the most depressed area, and width of the depression, will be recorded in the sagittal view on transvaginal ultrasound. Secondary endpoints will include total operative time, suture application time (from birth to the end of uterine suturing), operative blood loss, number of additional Z-sutures or continuous sutures required to stop bleeding, maternal abnormality frequency (surgical complications and postoperative infections), surgeon's years of experience, and clinical interpretation of individual subscale scores. DISCUSSION: This study shall provide important evidence on the optimal suture for preventing hysterotomy wound thinning after the first cesarean section. TRIAL REGISTRATION: National Institute of Public Health, Japan: jRCT1062200001 (May 7, 2020; https://rctportal.niph.go.jp/en/detail?trial_id=jRCT1062200001) and Okayama University Certified Review Board: CRB6180001 (April 9, 2020, version 3.0).

The assessment of the fetal heart function using two-dimensional speckle tracking with a high frame rate.

BACKGROUND: Two-dimensional (2D) speckle tracking echography (STE) is a new diagnostic modality that allows for the assessment of myocardial deformation. The present study assessed the feasibility of fetal 2D-STE at a high frame rate and determined the reference values for left ventricular (LV)- and right ventricular (RV)- global longitudinal strain (GLS). METHODS: In this prospective study, 109 fetuses with gestational ages ranging from 18 to 38 weeks underwent 2D echocardiography between August 2018 and December 2019. All recordings were performed using the Aplio i800 (CANON Medical Systems Corporation, Tochigi, Japan) and a convex probe (4 MHz) for fetuses. RESULTS: Longitudinal peak systolic strain measurements were obtained in 98 of 109 healthy fetuses (90%). All ultrasound videos were recorded with a median frame rate of 172 (range, 100-274) frames/s. The LV-GLS was almost constant, regardless of the number of weeks since the second trimester (r = -0.0087, p = ns). The RV-GLS decreased significantly with gestational age (r = 0.39, p < 0.01). CONCLUSIONS: STE may be useful for quantifying the systolic myocardial function in a fetus.

A case of subclinical Cushing's syndrome in pregnancy with superimposed preeclampsia.

When we see preexistent hypertension in pregnancy, subclinical Cushing's syndrome should be considered in the differential diagnosis since this disorder can cause perinatal complications. MRI can be useful for identifying adrenal incidental tumors during pregnancy.

Relationship between Intracellular Signaling of the (Pro)renin Receptor and the Pathogenesis of Preeclampsia.

An association between preeclampsia and (pro)renin was recently reported. Intracellular signaling of the (pro) renin receptor [(P)RR] increases the expressions of TGF-ß and PAI-1. In this study we sought to clarify the involvement of (pro)renin in the pathogenesis of preeclampsia via the intracellular signaling of (P)RR on preeclampsia placentas. Activated (pro)renin plasma concentrations were compared between pregnant women with (n=15) and without (n=28) preeclampsia. The placentas were immunohistochemically evaluated with anti-HIF-1α and anti-(P)RR antibodies. HTR-8/SVneo cells were cultured under hypoxic conditions and treated with human recombinant (pro)renin. The mRNA expressions of HIF-1α, (P)RR, PAI-1, TGF-ß, and ET-1 were also examined by real-time RCR. The activated (pro)renin plasma concentration was significantly higher in the third vs. the second trimester in the preeclampsia patients. HIF-1α and (P)RR expressions were significantly increased in the preeclampsia placentas. The mRNA expressions of PAI-1, TGF-ß, and ET-1 were significantly increased in the experiments using recombinant (pro)renin vs. hypoxic conditions. (P)RR expression in preeclampsia placentas is increased by persistent hypoxia through the second and third trimesters, and PAI-1, TGF-ß, and ET-1 production is increased via (P)RR. Our results suggest that ET-1 production via the intracellular signaling of (P)RR is important in the pathogenesis of preeclampsia.

Assessment of glucose kinetics with real-time continuous glucose monitoring during labor.

AIM: Changes in glucose levels during labor have not been sufficiently investigated in pregnant women. Using real-time continuous glucose monitoring, we aimed to assess glucose kinetics during labor among pregnant women with gestational diabetes mellitus (PwGDM), and those with normal glucose tolerance (PwNGT). METHODS: Japanese PwGDM and PwNGT who had planned a transvaginal delivery at Okayama University Hospital were enrolled. The correlation between changes in glucose levels during labor among the PwGDM and PwNGT groups at four time periods was assessed: (i) active phase of 1st stage of labor; (ii) 2nd stage of labor; (iii) postpartum 0-12 h; and (iv) postpartum 12-48 h. RESULTS: In total, 18 and 22 PwGDM and PwNGT, respectively, were enrolled. During labor, both groups had similar changes in glucose levels over time, which peaked during period 3. The main effect of glucose level changes was the labor period (P < 0.001), not the presence of gestational diabetes mellitus. Furthermore, differences in glucose levels in the PwGDM group were observed between periods 1 and 2 (P = 0.037), 1 and 3 (P = 0.024), 3 and 4 (P = 0.005); differences in glucose levels in the PwNGT group were observed between periods 3 and 4 (P = 0.024). CONCLUSION: During labor, both PwGDM and PwNGT groups showed similar changes in glucose levels over time. During delivery, the PwGDM who regularly measured their own glucose levels could be managed using the same nutritional management methods as those for PwNGT.

A Case of Nager Syndrome Diagnosed Before Birth.

Nager syndrome is a rare disease involving severe micrognathia and upper limb shortening. In this report, we describe a case in which micrognathia of the fetus was suspected based on the observation of upper limb shortening during detailed B mode and 3D/4D ultrasonographic observation, and combined fetal MRI and 3D-CT led to a prenatal diagnosis of Nager syndrome. Upon birth, because severe micrognathia caused airway obstruction and made it difficult to spread the larynx for intubation, effective ventilation could not be carried out and a tracheostomy was necessary. Since a differential diagnosis of Nager syndrome can be made based on the fact that micrognathia typically co-occurs with upper limb shortening, it is possible to diagnose the disease before birth and prepare for life-saving measures accordingly.

Successful Delivery after Abdominal Radical Trachelectomy, Using Transabdominal Cerclage in Early Pregnancy.

Radical trachelectomy (RT) is a fertility-sparing surgery for cervical cancer. Postoperative pregnancies have a high risk of abortion and prematurity. To prevent this, a procedure involving transabdominal cerclage (TAC) was devised for shortened cervical canals post-RT. Here we describe the successful management of a pregnancy after abdominal RT (ART). The 34-year-old patient was gravida 1, para 0. When she was 27, she underwent ART for stage Ib1 cervical cancer, and she became pregnant 7 years later. Because her cervical canal was 16.7 mm during early pregnancy, we performed TAC at 12 weeks of pregnancy. Post-surgery, we administered an infusion of ritodrine hydrochloride for tocolysis. A selective caesarean section was performed at 36 weeks, with the delivery of a healthy infant.

Upregulation of Angiogenic Factors via Protein Kinase C and Hypoxia-induced Factor-1α Pathways under High-glucose Conditions in the Placenta.

Abnormal glucose metabolism during pregnancy is an established risk factor for preeclampsia (PE). Disruption of the balance between placental angiogenic factors is linked to PE pathophysiology. We examined whether hypoxia-induced factor-1α (HIF-1α) and protein kinase Cß (PKCß) are involved in the regulation of placental angiogenic factors under high-glucose conditions in vitro. The human choriocarcinoma cell lines BeWo and JEG-3, and the human trophoblast cell line HTR-8/SVneo were cultured with 10 and 25 mmol/L glucose [control glucose group (CG) and high-glucose group (HG), respectively]. We examined the changes in HIF-1α, soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) expression in the CG and HG by real-time PCR and ELISA. PKC activation was also measured by ELISA. The expressions of HIF-1α, sFlt-1, PlGF, and VEGF were significantly higher in the HG than in the CG. PKC activity was significantly increased in the HG. High glucose affected the expression of angiogenic factors in choriocarcinoma cells via the PKCß and HIF-1α pathways, suggesting their involvement in PE pathogenesis.

Assessment of resting energy expenditure and body composition in Japanese pregnant women with diabetes.

AIMS/INTRODUCTION: To measure longitudinal changes in resting energy expenditure and body composition of Japanese pregnant women with or without diabetes. MATERIALS AND METHODS: The study population consisted of women who had delivered a live singleton neonate after 22 weeks' gestation at Okayama University Hospital from July 2013 to June 2017. Resting energy expenditure and body composition were measured in the first trimester, second trimester, third trimester and postpartum. RESULTS: A total of 144 women participated in this study: 103 with normal glucose tolerance and 41 with diabetes. The resting energy expenditure (kcal/day) of pregnant women with normal glucose tolerance was significantly higher in the third trimester (1,644 ± 234) than in the first (1,461 ± 215) and second trimesters (1,491 ± 219), and postpartum (1,419 ± 254), whereas that of pregnant women with diabetes did not significantly change during all periods (1,568 ± 404, 1,710 ± 332, 1,716 ± 251, 1,567 ± 249). The resting energy expenditure of women with good glycemic control was lower than that of women with poor control. Fat-free mass was closely correlated with resting energy expenditure. CONCLUSIONS: The resting energy expenditure of Japanese pregnant women with normal glucose tolerance was significantly increased in the third trimester. The resting energy expenditure of women with good glycemic control was lower than that of women with poor control. Resting energy expenditure and fat-free mass are potential indexes for medical nutrition therapy in pregnant women with diabetes.