Amniotic sac reference interval in early pregnancy between 7 and 10 weeks' gestation.

OBJECTIVE: To establish a normal reference interval for amniotic sac measurements between 7 and 10 weeks of gestation and its relative size in relation to the gestational sac and the embryo. METHOD: This was a prospective, cross-sectional study of consecutive women presenting to UCLH Early Pregnancy Unit between August 2022 to June 2023. We included live, normally sited, singleton pregnancies with a normal 20-week anomaly scan. We collected 120 cases per gestational week totaling 360 cases. We performed an inter and intra-observer variability assessment in the measurement of mean ASD in 30 patients. Regression analyses were used to establish reference intervals for GSD to CRL, ASD to CRL, GSD to ASD and GSD:ASD ratio to CRL. The fitted regression line was calculated, along with a 90% prediction interval and the R2 value. RESULTS: There was good interobserver agreement (difference 0.007mm ± 1.105 (95%CI -2.160 to 2.174)) and good intra-observer agreement between Observer A (0.007 ± 1.105 (-2.160 to 2.174)) and Observer B (-0.014 ± 0.919 (-1.814 to 1.786)) in the measurement of mean ASD in 30 patients. Regression analyses showed a highly statistically significant association between each pair of values (all p-values <0.001). There were significant quadratic associations between mean GSD and CRL (R2 = 56%) and mean GSD and ASD (R2 = 60), significant cubic association between ASD and CRL (R2 = 90%) and significant quadratic association between GSD to ASD ratio and CRL (R2 = 68%). The regression equations were used to quantify the values of ASD and GSD to ASD ratios for a range of CRL values and gestational age in days. CONCLUSION: Our study has produced comprehensive reference intervals for amniotic sac size in early pregnancy which could be used in routine clinical practice. This article is protected by copyright. All rights reserved.

Ultrasound study of natural progression of ovarian endometriomas.

OBJECTIVES: The aim of this study was to determine the natural history of ovarian endometriomas in women who are managed expectantly. METHODS: This was a retrospective cohort study of 83 women with evidence of ovarian endometriomas, who were managed expectantly between April 2007 to May 2022. The study was conducted in the Department of Women's Health, University College London Hospitals and The Gynecology Ultrasound Centre, London, UK. We searched our ultrasound clinic databases to identify women aged 18 years or older with evidence of ovarian endometriomas that were managed expectantly for ≥ 6 months. All women attended for a minimum of two ultrasound scans by a single expert ultrasound operator. In addition to patient demographics, we recorded the number, mean diameter and location of each cyst. The cyst growth rate was expressed as annual change in the mean diameter. RESULTS: 1,922 women attended our gynecology clinic during the study period who were found to have evidence of moderate or severe endometriosis on pelvic ultrasound examination. A total of 83 women had evidence of ovarian endometriomas and were managed expectantly. The median age of women was 39 (range 26 - 51). Each woman had at least two ultrasound scans performed by a single expert operator at a minimum interval of ≥6 months. 50/83 (60%, 95% CI 49-71) women had single cysts and the remainder had multiple cysts. The median number of endometriomas per patient was 1 (range 1 - 5) and the median follow up time was 634 days (range 187 - 2984). 39/83 (47%, 95% CI 36 - 58) women experienced an overall reduction in size of cysts, in 18/83 (22%, 95% CI 13 - 32) the cysts increased in size and in 26/83 (31% 95% CI 22 - 42) women, no meaningful change was observed. The median change in mean diameter of cysts per woman during the study period was -2.7 mm (-57.7 - +39.3), with an annual growth rate of -1.7 mm/year/woman (-24.6 - +42.0). Overall, cysts were smaller at the follow up visit [median diameter 22.3mm (6.7 - 77) vs. 18.5mm (5 - 72) p = 0.009]. We did not identify any clinical characteristics that could reliably predict the chance of endometrioma progression. CONCLUSION: In the majority of women with ultrasound diagnosis of ovarian endometriomas, the cysts do not increase in size significantly over time and they could be managed expectantly. This evidence may help clinicians when counselling asymptomatic or minimally symptomatic women about the options to manage their ovarian endometriomas. This article is protected by copyright. All rights reserved.

Natural history of endometriosis in pregnancy: ultrasound study of morphology of deep endometriosis and ovarian endometrioma.

OBJECTIVE: To assess the morphological appearance of deep endometriosis and ovarian endometrioma in pregnancy using pelvic ultrasound examination. METHODS: This was a prospective observational cohort study conducted over 3 years at University College London Hospital, which is a tertiary level referral unit for early pregnancy complications and an accredited endometriosis center. All women who participated provided written consent and were invited for surveillance ultrasound examination at the time of their routine scans in pregnancy. All scans were performed by a single operator to eliminate interobserver variability. The change in size of ovarian endometrioma and nodules was reported as change in their mean diameter. Ovarian endometrioma with irregular thick inner walls, hyperechoic papillary projections and/or high vascularity and hyperechoic nodules with moderate to high vascularity were reported as decidualized. RESULTS: Sixty-five women with a live, normally sited pregnancy and concomitant ultrasound features of deep and/or ovarian endometriosis were included in the study. The median age of the study population was 34 (range, 23-44) years, and the median gestational age at presentation was 7 + 6 (range, 3 + 6 to 18 + 0) weeks. From the cohort, 47/65 (72%) were nulliparous, 48/65 (74%) had a previous diagnosis of endometriosis and 19/65 (29%) conceived via in-vitro fertilization. There were 10/65 (15% (95% CI, 7-24%)) women with ovarian endometrioma alone, 28/65 (43% (95% CI, 31-55%)) with endometriotic nodules alone and the remaining 27/65 (42% (95% CI, 30-54%)) had both. Of the women with ovarian endometrioma who underwent follow-up, 29/34 (85% (95% CI, 73-97%)) experienced cyst regression, 2/34 (6% (95% CI, 0-14%)) experienced cyst growth, and in 3/34 (9% (95% CI, 0.0-18%)) women, cyst size was unchanged. In 10/34 (29% (95% CI, 14-45%)), there was complete resolution of all cysts. Of the women with nodules who underwent follow-up, 43/51 (84% (95% CI, 74-94%)) experienced nodule regression, 2/51 (4% (95% CI, 0-9%)) experienced nodule growth and, in 6/51 (12% (95% CI, 3-21%)) women, nodule size was unchanged. In 4/51 (8% (95% CI, 0-15%)) women, there was complete resolution of all nodules. In 5/37 (14% (95% CI, 3-25%)) women who attended postnatal follow-up, complete resolution of all endometriotic lesions occurred during pregnancy. In 10/34 (29% (95% CI, 14-45%)) women with ovarian endometrioma and 27/51 (53% (95% CI, 39-67%)) women with nodules, a pattern of growth was observed in the first and second trimesters, followed by regression later in pregnancy. Features of decidualization were observed in 17/34 (50% (95% CI, 33-67%)) women with ovarian endometrioma, most commonly in the first trimester, and in 25/51 (49% (95% CI, 35-63%)) women with nodules, most commonly in the second trimester. CONCLUSIONS: For the majority of women, despite features of decidualization being common in the first and second trimesters, ovarian endometrioma and deep nodules regress during pregnancy. Morphological changes of endometriosis in pregnancy are difficult to differentiate from characteristics of malignant lesions. Better understanding of the appearance of endometriosis in pregnancy is vital to minimize intervention and help counsel women regarding their condition. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Reproductive outcome in 326 women with unicornuate uterus.

OBJECTIVES: To study the reproductive outcomes of women with a unicornuate uterus and compare them to those of women with no congenital uterine anomaly. METHODS: This was a single-center, retrospective cohort study. Cases were women aged at least 16 years who were diagnosed with a unicornuate uterus on transvaginal/transrectal ultrasound between January 2008 and September 2021. Controls were women with no congenital uterine anomaly matched 1:1 by age and body mass index. The primary outcome was live-birth rate. Secondary outcomes were pregnancy loss (miscarriage, ectopic pregnancy, termination of pregnancy), preterm delivery, mode of delivery and concomitant gynecological abnormalities (endometriosis, adenomyosis, fibroids). RESULTS: Included in the study were 326 cases and 326 controls. Women with a unicornuate uterus had a significantly lower live-birth rate (184/388 (47.4%) vs 229/396 (57.8%); P = 0.004) and higher rates of overall miscarriage (178/424 (42.0%) vs 155/465 (33.3%); adjusted odds ratio (aOR), 2.21 (95% CI, 1.42-3.42), P < 0.001), ectopic pregnancy (26/424 (6.1%) vs 11/465 (2.4%); aOR, 2.52 (95% CI, 1.22-5.22), P = 0.01), preterm delivery (45/184 (24.5%) vs 17/229 (7.4%); aOR, 3.04 (95% CI, 1.52-5.97), P = 0.001) and Cesarean delivery (116/184 (63.0%) vs 70/229 (30.6%); aOR, 2.54 (95% CI, 1.67-3.88), P < 0.001). Rudimentary-horn pregnancies accounted for 7/26 (26.9%) ectopic pregnancies in the study group. Women with a unicornuate uterus were more likely to have endometriosis (17.5% vs 10.7%; P = 0.018) and adenomyosis (26.7% vs 15.6%; P = 0.001), but were not more likely to have fibroids compared with controls. Women with a functional rudimentary horn were more likely to have pelvic endometriosis compared to those without (odds ratio, 2.4 (95% CI, 1.4-4.1), P = 0.002). CONCLUSIONS: Pregnant women with a unicornuate uterus should be classified as high risk. Removal of a functional rudimentary horn should be discussed with the patient to prevent a rudimentary-horn ectopic pregnancy. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Imaging in gynecological disease (18): clinical and ultrasound characteristics of urinary bladder malignancies.

OBJECTIVE: To describe the clinical and ultrasound characteristics of urinary bladder malignancies diagnosed on transvaginal ultrasound in women presenting with suspected gynecological problems. METHODS: This was a multicenter retrospective study of women with a histological diagnosis of urinary bladder malignancy that was suspected on transvaginal ultrasound examination. The cases were collected from three centers that specialize in the use of pelvic ultrasound and had been examined between January 2007 and October 2018. Clinical data were obtained from the computer databases and all tumor images were assessed by two of the authors (D.J. and J.K.) to identify characteristic sonographic patterns. We compared the characteristics of tumors between women presenting with symptoms suspicious of urinary bladder malignancy and those without such symptoms. RESULTS: Thirty women with a confirmed diagnosis of urinary bladder malignancy on histological examination were included. Median age at diagnosis was 70.5 (range 36-88) years. The most common presenting symptom was postmenopausal bleeding, which was recorded in 18 (60%) women. Ten (33%) women had symptoms suspicious of bladder malignancy, of whom six had unexplained visible hematuria, three had unexplained recurrent urinary tract infections and one had dysuria and microhematuria. On histological examination, 23 (77%) women were diagnosed with primary bladder malignancy whilst seven (23%) had metastases in the bladder from other primary tumors. Out of 23 primary tumors, 21 (91%) were of urothelial origin (12 low grade and nine high grade). Most low-grade urothelial carcinomas appeared on ultrasound as irregular papillary growth (11/12, 92%) and were moderately to highly vascular on color Doppler examination (8/12, 67%). The ultrasound appearances of primary non-urothelial and metastatic tumors varied, without a clear common morphological tumor pattern. The tumors found in women with symptoms suggestive of bladder malignancy did not differ unequivocally from those detected in other women in terms of size, ultrasound morphology, vascularity or histological type. CONCLUSION: Urinary bladder malignancies can be detected in patients undergoing transvaginal ultrasound examination for suspected gynecological problems. Primary urothelial cancers have a relatively uniform morphological pattern, whilst the appearances of other bladder malignancies are more variable. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Predicted luteal phase length has no influence on success of vitrified-warmed blastocyst transfer in natural cycle.

BACKGROUND: This study evaluated the influence of menstrual cycle length, menstrual cycle variability and predicted luteal phase length on the success of vitrified-warmed blastocyst transfer in natural menstrual cycle using progesterone for luteal phase supplementation. METHODS: Consecutive women undergoing vitrified-warmed blastocyst transfer in natural menstrual cycle between January 2013 and December 2015 were included in this retrospective study. Patients' characteristics, clinical data and data about menstrual cycle length in the last year were collected from our database. Predicted luteal phase length (LPL) was defined as the period starting at ovulation (one day after positive urinary LH test) and ending on the last day before predicted menses, based on women's usual, minimal and maximal menstrual cycle length data. Logistic regression was used to identify the predictors significantly associated with live-birth. RESULTS: A total of 1195 FETs (frozen-thawed embryo transfers) resulted in 457 (38.24%) clinical pregnancies, 82 (17.94%), miscarriages and 371 live births (31.04%). There were no statistically significant differences in menstrual cycle length, menstrual cycle variability, day of LH surge, day of FET and predicted LPL between FET cycles resulting in live birth and those not resulting in live birth. In the multivariate logistic regression model, only women's age (OR 0.93, 95% CI: 0.90-0.96), transfer of morphologically optimal blastocysts (OR 2.17, 95% CI: 1.59-2.94) and endometrium thickness (OR 1.10, 95% CI: 1.03-1.17) were important independent prognostic factors for live birth. CONCLUSION: Menstrual cycle length, menstrual cycle variability and predicted LPL do not seem to be an important factor influencing live birth after FET in natural cycles with progesterone supplementation. Results of our study suggest that FET should not be cancelled if LH surge is detected before or after the predicted period in natural cycle with progesterone supplementation.

ESHRE/ESGE female genital tract anomalies classification system-the potential impact of discarding arcuate uterus on clinical practice.

STUDY QUESTION: What would be a potential impact of implementing the new ESHRE/European Society of Gynaecological Endoscopy (ESGE) female genital anomalies classification system on the management of women with previous diagnosis of arcuate uteri based on the modified American Society for Reproductive Medicine (ASRM) criteria? SUMMARY ANSWER: A significant number of women with previous diagnosis of arcuate uteri are reclassified as having partial septate uteri according to the new ESHRE/ESGE classification system which may increase the number of remedial surgical procedures. WHAT IS KNOWN ALREADY: The ESHRE/ESGE classification system has defined measurement techniques, reference points and specific cut-offs to facilitate the differentiation between normal and septate uteri. These criteria have been arbitrarily defined and they rely on the measurement of uterine wall thickness and depth of distortion of uterine fundus. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study. We searched our ultrasound clinic database from January 2011 to December 2014 to identify all women diagnosed with arcuate uterus on three-dimensional ultrasound according to the modified ASRM criteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: For each woman, the ultrasound images were stored in our clinical database and they were re-examined according to ESHRE/ESGE specifications. The presence and location of all acquired uterine anomalies, such as fibroids or adenomyosis was noted. We applied the two diagnostic approaches as specified by the ESHRE/ESGE classification: the main option (MO) and the alternative option (AO). We used the Kappa statistic to quantify the agreement between the two approaches. We also compared the number of previous miscarriages in women with normal and partial septate uteri according to the ESHRE/ESGE classification. Non-parametric Mann-Whitney and Kruskal-Wallis tests were used for the analyses and receiver-operating characteristic curves were constructed to assess the predictive values of the calculated uterine distortion indices for the detection of women at risk of suffering multiple pregnancy losses. MAIN RESULTS AND THE ROLE OF CHANCE: We included 270 women diagnosed with arcuate uterus in the study. In all, 77 women (28.5%, 95% confidence interval (CI) 23.1-33.9) had evidence of fibroids or adenomyosis. These abnormalities precluded the application of either proposed ESHRE/ESGE techniques to assess uterine morphology in 25 women (9.3%, 95% CI 5.8-12.7). When using the MO, 138/237 (58.2%, 95% CI 51.9-64.3) women were diagnosed with partial septate uterus compared to 61/230 (26.5%, 95% CI 21.2-32.6) women when using the AO. In 222 women in whom we were able to apply both MO and AO, there was agreement in the diagnosis of septate uterus between the two techniques in 146/222 cases (65.8%, 95% CI 59.3-71.7; Kappa 0.42, 95%CI 0.35-0.5). There was no statistical difference in the proportion of women with history of previous multiple miscarriages between those diagnosed with normal or partial septate uteri using either MO (6.2%, 95% CI 2.9-12.9 vs. 9.5%, 95% CI 5.6-15.6; P = 0.47) or AO (7.2%, 95% CI 4.2-12.1 vs. 11.7%, 95% CI 5.8-22.2; P = 0.29). LIMITATIONS, REASONS FOR CAUTION: This study was retrospective in nature and the definition of arcuate uterus used in the study is not universally accepted. The reproductive history data were collected retrospectively and therefore may be prone to bias. WIDER IMPLICATIONS OF THE FINDINGS: There are methodological weaknesses in the new ESHRE/ESGE classification system which would need to be addressed in future revisions. There was no significant difference in the past reproductive outcomes between women diagnosed with normal and anomalous uteri and the clinicians should exercise caution when offering surgical correction to women diagnosed with partial septate uteri using the new ESHRE/ESGE classification. STUDY FUNDING/COMPETING INTEREST(S): No study funding was received and no competing interests are present. TRIAL REGISTRATION NUMBER: N/A.

Surgical treatment of Cesarean scar ectopic pregnancy: efficacy and safety of ultrasound-guided suction curettage.

OBJECTIVES: To assess the efficacy of ultrasound-guided suction curettage for management of pregnancies implanted into the lower uterine segment Cesarean section scar. METHODS: This was a retrospective study including women diagnosed with Cesarean section scar pregnancy at two large tertiary referral early pregnancy units between 1997 and 2014. Surgical evacuation was offered to selected women presenting in the first trimester ≤ 14 weeks' gestation. All procedures were performed transcervically under ultrasound guidance using suction curettage. A modified Shirodkar cervical suture was used in women who required additional measures to secure hemostasis. RESULTS: A total of 232 women with Cesarean section scar pregnancy were seen at the referral units; 191/232 (82.3%) women were treated surgically. The median intraoperative blood loss was 100 mL (range, 10-3000 mL); 9/191 (4.7% (95% CI, 1.7-7.7%)) women required blood transfusion and, in one (0.5% (95% CI, 0-1.5%)), life-saving hysterectomy had to be performed because of uncontrollable intraoperative bleeding. Of the women who attended for follow-up, 7/116 (6.0% (95% CI, 1.7-10.3%)) required a repeat surgical procedure because of retained products of conception. Multivariable analysis showed that the gestational sac diameter (odds ratio (OR), 1.10 (95% CI, 1.03-1.17)) and pregnancy vascularity on Doppler examination (OR, 3.41 (95% CI, 1.39-8.33)) were significant predictors of heavy intraoperative blood loss (> 1000 mL). CONCLUSIONS: Ultrasound-guided suction curettage is an effective method for the treatment of pregnancies implanted into a lower uterine segment Cesarean section scar and is associated with a low risk of blood transfusion and hysterectomy. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Should a detailed ultrasound examination of the complete urinary tract be routinely performed in women with suspected pelvic endometriosis?

STUDY QUESTION: Is there any benefit to including the routine examination by ultrasound of the bladder, ureters and kidneys of women with endometriosis? SUMMARY ANSWER: The benefit of examination of the complete urinary tract of women with suspected endometriosis is that ureteric endometriosis, with or without hydronephrosis, can be detected which facilitates early intervention to prevent nephropathy. WHAT IS ALREADY KNOWN: Women with endometriosis can get ureteric obstruction but there is no clear consensus on the correct diagnostic technique. Ultrasound is accurate at detecting women with bladder endometriosis but ureteric involvement has not been assessed previously. STUDY DESIGN, SIZE, DURATION: This was a prospective observational study, conducted at a teaching hospital over a period of 14 months. A total of 848 women presenting with chronic pelvic pain were included into the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women with chronic pelvic pain underwent a detailed transvaginal and transabdominal pelvic ultrasound examination to investigate possible causes of their symptoms. This included a systematic assessment of the urinary bladder, pelvic sections of the ureters and kidneys. The ultrasound findings were compared with findings at surgery and the results of targeted urological imaging and interventions. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 848 women presenting with chronic pelvic pain were included into the study. 28/848 women (3.3% 95% CI 2.1-4.5) had evidence of urinary tract abnormalities on initial ultrasound scan. Among these 17/848 (2.0% 95% CI 1.06-2.94) had evidence of urinary tract endometriosis, whilst 11/848 (1.3% 95% CI 0.54-2.06) women had other urinary tract abnormalities. Among women with urinary tract endometriosis 11/17 (65%) had evidence of ureteric involvement, 3/17 (18%) had both ureteric and bladder disease and 3/17 (18%) had bladder disease only. 12/17 (59%) women with urinary tract endometriosis also had evidence of hydronephrosis. The diagnosis of ureteral endometriosis had a sensitivity of 12/13 (92%) (95% CI 63.9-99.8), specificity 151/151 100% (95% CI 97.6-100), PPV 100% (95% CI 73.5-100), NPV 99.3% (95% CI 96.3-99.9%) LR- 0.08 (95% CI 0.01-0.39). LIMITATIONS, REASONS FOR CAUTION: The routine examination of the complete urinary tract including the distal ureters is a novel technique that should be evaluated in different populations. WIDER IMPLICATIONS OF THE FINDINGS: Ultrasound is an accurate test to diagnose urinary tract involvement in women with suspected pelvic endometriosis and examination of the complete urinary tract should become an integral part of ultrasound assessment of women with suspected endometriosis.

Should an intrauterine insemination with donor semen be performed 1 or 2 days after the spontaneous LH rise? A prospective RCT.

STUDY QUESTION: What is the impact on pregnancy rates when intrauterine insemination (IUI) is performed 1 or 2 days after the spontaneous LH rise? SUMMARY ANSWER: IUI 1 day after the spontaneous LH rise results in significantly higher clinical pregnancy rates compared with IUI performed 2 days after the LH rise. WHAT IS KNOWN ALREADY: IUI is scheduled within a limited time interval during which successful conception can be expected. Data about the optimal timing of IUI are based on inseminations following ovarian stimulation. There is no available evidence regarding the correct timing of IUI in a natural menstrual cycle following the occurrence of a spontaneous LH rise. STUDY DESIGN, SIZE, DURATION: A prospective RCT, including patients undergoing IUI with donor sperm in a natural menstrual cycle. IUI cycles (n = 435) were randomized between October 2010 and April 2013, of which 23 were excluded owing to protocol deviation and 412 received the allocated intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serial serum LH concentrations were analysed in samples taken between 07:00 and 09:00 h to detect an LH rise from Day 11 of the cycle onwards. The subjects were randomized to receive insemination either 1 or 2 days after the observed LH rise. In the final analysis, there were 213 cycles in the group receiving IUI 1 day after the LH rise and 199 cycles in the group receiving IUI 2 days after the LH rise. MAIN RESULTS AND THE ROLE OF CHANCE: Significantly higher clinical pregnancy rates per IUI cycle were observed in patients undergoing IUI 1 day after the LH rise when compared with patients undergoing IUI 2 days after the LH rise [19.7 (42/213) versus 11.1% (22/199), P = 0.02]. In view of the timing of sampling for LH, the inseminations were performed at 27 h (±2 h) and 51 h (±2 h) after detection of the LH rise. The risk ratio of achieving a clinical pregnancy if IUI was scheduled 1 day after the LH rise compared with 2 days was 1.78 [95% confidence interval (CI), 1.11-2.88]. This points towards a gain of one additional clinical pregnancy for every 12 cycles performed 1 day instead of 2 days after the LH rise. When analysing the results per patient, including only women who underwent their first treatment cycle of insemination, the outcome was in line with the per cycle analysis, demonstrating an 8% difference in pregnancy rate in favour of the early group (20.5 versus 12.2%), however, this difference was not significant. LIMITATIONS, REASONS FOR CAUTION: Optimal monitoring for the occurrence of the LH rise involves several daily LH measurements, which is not always amenable to everyday clinical practice, however, daily sampling was sufficient to detect a significant difference in pregnancy rate. The strict inclusion of a highly selected population of patients who underwent IUI in a natural cycle may have been a limitation. IUI in a natural menstrual cycle confers lower success rates compared with IUI following ovarian stimulation and is not suitable for patients with ovulatory dysfunction. Furthermore, a similar study in a larger number of women is required to confirm the result in terms of pregnancy rate per patient. WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT to show that timing of IUI in a natural menstrual cycle is important and that IUI should be performed 1 day after the LH rise, rather than 2 days post-LH rise. Daily monitoring of the rise in LH, as performed in our study, can be adopted to achieve a higher pregnancy rate per IUI cycle. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. All authors declare to have no conflict of interest with regard to this trial. TRIAL REGISTRATION NUMBER: The trial was registered at clinicaltrials.gov (NCT01622023).

A single amino acid substitution in herpes simplex virus type 1 VP16 inhibits binding to the virion host shutoff protein and is incompatible with virus growth.

In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAdeltaSma showed that VP16(L344A) was able to complement the growth of 8MAdeltaSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

Comparative efficiencies of C-terminal signals of native glycophosphatidylinositol (GPI)-anchored proproteins in conferring GPI-anchoring.

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post-translational modification has a C-terminal GPI-anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed omega-->omega + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C-terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3' end-sequence encoding omega-2 to the C-terminus with the corresponding C-terminal sequences of nine other human GPI-anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS-PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out-performing the others in GPI-anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at omega-->omega + 2.

Partial liquid ventilation combined with kinetic therapy in acute respiratory failure in piglets.

OBJECTIVE: To investigate the effect of the combination of kinetic therapy (KT) with partial liquid ventilation (PLV) on gas exchange, lung mechanics and hemodynamics in acute lung injury (ALI). DESIGN: Prospective, randomized, controlled pilot study. SETTING: University research laboratory. SUBJECTS: Eleven piglets weighing 8.3+/-0.9 kg. INTERVENTION: ALI was induced by the infusion of oleic acid (0.08 ml/kg) and repeated lung lavages with 0.9% NaCl (20 ml kg(-1)). Thereafter the animals were randomly assigned either for PLV or a combination of PLV with KT (PLV/KT). The dose of perfluorocarbon administered was 30 ml/kg, evaporative losses were substituted with 5 ml/kg per h. MEASUREMENTS AND MAIN RESULTS: Airway pressures, tidal volumes, dynamic compliance (Cdyn), expiratory airway resistance and arterial blood gases were measured. Hemodynamic monitoring included right atrial, mean pulmonary artery, pulmonary capillary wedge and mean systemic arterial pressures, and continuous flow recording of the pulmonary artery. In both groups the induction of ALI significantly reduced PaO2/FIO2 Cdyn and cardiac output, and significantly increased pulmonary artery pressure. After the initiation of PLV there was a significant increase of PaO2/FIO2, and Cdyn, and a significant decrease of pulmonary artery pressure in both groups. Except the PaCO2, which showed significantly lower values in the PLV/KT group, no variables showed any differences between the two groups. CONCLUSION: The additional use of KT did not show beneficial effects on oxygenation and lung mechanics during PLV. However, at constant minute ventilation PaCO2 levels were significantly lower during PLV/KT, indicating some positive influence on the ventilation/perfusion distribution within the lung. Extreme body positions during PLV/KT did not show any significant hemodynamic side effects.

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5' extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans approximately 25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16.

The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional assembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.

Mammalian glycosylphosphatidylinositol-anchored proteins and intracellular precursors.

Glycosylphosphatidylinositol-anchored proteins can be specifically identified by several methods. PI-PLC digestion analyses, the most widely used technique, can be performed more reliably when conducted with purified protein and phase partitioning to exclude steric effects and when combined with alkaline hydrolysis to control for inositol acylation. Reductive radiomethylation not only can definitively identify a candidate protein as being GPI anchored, but also can provide information on the number of amine components (GlcN, ethanolamine) in the anchor structure. Biosynthetic labeling with anchor precursors is relatively specific when performed with [3H]ethanolamine or [3H]inositol. Incorporation of the precursors additionally can be used to (1) document anchor transfer to primary translation products, (2) identify soluble derivatives of GPI-anchored proteins that have been released from cell surfaces, and (3) localize the site of GPI anchor attachment within a GPI-anchored protein. A pathway for mammalian GP anchor assembly is depicted in Fig. 12. Initially GlcNAc is transferred to PI. The resulting GlcNAc-PI is then deacetylated to yield GlcN-PI. After that step, several points of divergence are identifiable between the mammalian and T. brucei pathways: (1) all mammalian Man-containing intermediates are built on acylated inositol phospholipids; (2) a proximal phosphoethanolamine is found in mammalian GPI anchor intermediates and is added to Man 1 prior to incorporation of Man 2 and Man 3; (3) no Gal branching substituent is added to the mammalian core glycan; and (4) the most polar mammalian GPI contains a third phosphoethanolamine substituent linked to the 6 position of Man 2.

Stimulation of glycogen synthesis by insulin in human erythroleukemia cells requires the synthesis of glycosyl-phosphatidylinositol.

Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.