Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells.

INTRODUCTION: Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. METHODS: Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. RESULTS: BaP (0.05-1µM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. DISCUSSION: First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

Eomesodermin, HAND1, and CSH1 proteins are induced by cellular stress in a stress-activated protein kinase-dependent manner.

Eomesodermin (Eomes) is a transcription factor essential for trophoblast development. Stress stimuli activate stress-activated protein kinase (MAPK8/9) and modulate transcription factors in trophoblast stem cells (TSC). In this study, we test the hypothesis that stress-induced Eomes upregulation and downstream trophoblast development are MAPK8/9-dependent. Immunocytochemical and immunoblot assays suggest that Eomes is induced by hyperosmolar stress in a dose- and time-dependent manner. Two MAPK8/9 inhibitors that work by different mechanisms, LJNKl1 and SP600125, block induction of Eomes protein by stress. During normal TSC differentiation, the transcription factor heart and neural crest derivatives expressed 1 (HAND1) is dependent on Eomes, and chorionic somatomammotropin hormone 1 (CSH1) expression is dependent on HAND1. Similar to Eomes, HAND1 and CSH1 induction by stress are MAPK8/9-dependent, and CSH1 is induced in nearly all stressed TSC. CSH1 induction normally requires downregulation of the transcription factor inhibitor of differentiation 2 (ID2) as well as HAND1 upregulation. It was shown previously that hyperosmolar stress induces AMP-activated protein kinase (PRKAA1/2)-dependent ID2 loss in a MAPK8/9-independent manner. Inhibition of PRKAA1/2 with compound C and LJNKl1, more than MAPK8/9 inhibitors alone, inhibits the induction of CSH1 by stress. Taken together these data suggest that stress-induced MAPK8/9 and PRKAA1/2 regulate transcription factors Eomes/HAND1 and ID2, respectively. Together this network mediates induction of CSH1 by stress. Therefore, stress triggers a proportional increase in a normal early TSC differentiation event that could be adaptive in inducing CSH1. But the flexibility of TSC to undergo stress-induced differentiation could lead to pathophysiological consequences if stress endured and TSC differentiation became unbalanced.

Interpreting the stress response of early mammalian embryos and their stem cells.

This review analyzes and interprets the normal, pathogenic, and pathophysiological roles of stress and stress enzymes in mammalian development. Emerging data suggest that stem cells from early embryos are induced by stress to perform stress-enzyme-mediated responses that use the strategies of compensatory, prioritized, and reversible differentiation. These strategies have been optimized during evolution and in turn have aspects of energy conservation during stress that optimize and maximize the efficacy of the stress response. It is likely that different types of stem cells have varying degrees of flexibility in mediating compensatory and prioritized differentiation. The significance of this analysis and interpretation is that it will serve as a foundation for yielding tools for diagnosing, understanding normal and pathophysiological mechanisms, and providing methods for managing stress enzymes to improve short- and long-term reproductive outcomes.

Cellular stress causes reversible, PRKAA1/2-, and proteasome-dependent ID2 protein loss in trophoblast stem cells.

Stress reduces fertility, but the mechanisms mediating this are not understood. For a successful pregnancy, placental trophoblast stem cells (TSCs) in the implanting embryo proliferate and then a subpopulation differentiates to produce hormones. Normally, differentiation occurs when inhibitor of differentiation 2 (ID2) protein is lost in human and mouse placental stem cells. We hypothesize that stress enzyme-dependent differentiation occurs in association with insufficient TSC accumulation. We studied a well-defined model where TSC differentiation requires ID2 loss. The loss of ID2 derepresses the promoter of chorionic somatomammotropin hormone 1 (CSH1), the first hormone after implantation. Csh1 mRNA is known to be induced in stressed TSCs. In this study, we demonstrate that AMP-activated protein kinase (PRKAA1/2, aka AMPK) mediates the stress-induced proteasome-dependent loss of ID2 at high stress levels. At very low stress levels, PRKAA1/2 mediates metabolic adaptation exemplified by the inactivation of acetyl coA carboxylase by phosphorylation without ID2 loss. At the highest stress levels, irreversible TSC differentiation as defined by ID2 loss and slower cell accumulation occurs. However, lower stress levels lead to reversible differentiation accompanied by metabolic adaptation. These data support the hypothesis that PRKAA1/2 mediates preparation for differentiation that is induced by stress at levels where a significant decrease in cell accumulation occurs. This supports the interpretation that enzyme-mediated increases in differentiation may compensate when insufficient numbers of stem cells accumulate.

Lemierre's syndrome complicating pregnancy.

Lemierre's syndrome is an anaerobic suppurative thrombophlebitis involving the internal jugular vein secondary to oropharyngeal infection. There is only one previous case report in pregnancy which was complicated by premature delivery of an infant that suffered significant neurological damage. We present an atypical case diagnosed in the second trimester with a live birth at term. By reporting this case, we hope to increase the awareness of obstetricians to the possibility of Lemierre's syndrome when patients present with signs of unabating oropharyngeal infection and pulmonary symptoms.

A case of 46,X,der(X)t(X;X)(q22.1;p11) Xq22.1-->Xqter in a 12-year-old girl with premature ovarian failure.

Premature ovarian failure due to Xp duplication and Xq deletion has been reported in four patients, the youngest of whom was 18 years old. The diagnosis has been made with new techniques for genetic analysis, such as comparative genomic hybridization and fluorescence in situ hybridization. We report the youngest case (a 12-year-old who presented with irregular menses), of premature ovarian failure due to Xp duplication and Xq deletion and the first with 46,X,der(X)t(X;X)(q22.1;p11). The diagnosis was made using C-banding and fluorescent in situ hybridization with locus-specific probes. This case highlights the need to use advanced genetic strategies to determine karyotypic and phenotypic abnormalities.

Abdominal packing for intractable obstetrical and gynecologic hemorrhage.

Hemorrhage continues to be a serious complication of both obstetrical and gynecologic surgeries. Physicians have used packing in cases of uncontrollable hemorrhage for many years, and this article reports on a modification of standard packing techniques that prevents some of the limitations of traditional packing. This technique was used in 1 patient after cesarean hysterectomy and 3 patients after debulking surgery for advanced gynecologic cancer. The pack consists of a wide piece of ribbon gauze and a Penrose drain. One end of the ribbon gauze is draped over a layer of surgicel (oxidized regenerated cellulose), while the rest is threaded through a 1-inch Penrose drain tightly folded several times to maintain pressure over the bleeding area. The other end of the Penrose drain, with the ribbon gauze visible within it, is inserted through a stab incision in the ipsilateral side of the lower abdomen. This technique allows for continuous bleeding assessment and easy removal of the gauze at bedside.

Critical appraisal workshops to promote evidence-based healthcare.

We evaluated the effect of literature appraisal workshops on participants views, attitudes and knowledge about evidence-based medicine in the West Midlands region in 1998. The performance of 55 practitioners was evaluated, before and after attending the workshop. After attending the workshop, participants paid more attention to the study design (81% vs. 98%, P=0.02), they did not find research evidence confusing (35% vs. 52%, P=0.05), and they felt more confident in assessing research evidence (26% vs. 59%, P=0.0001). Their mean knowledge scores improved from 47.3 (SD12.2) to 57.9 (SD 9.0) ( P=0.0001). Our critical appraisal skills workshops improved attitudes and knowledge needed for the provision of evidence-supported healthcare. Such workshops should be incorporated in postgraduate obstetrics and gynaecology training programmes.

Is waiting for an endogenous luteinizing hormone surge and/or administration of human chorionic gonadotrophin of benefit in intrauterine insemination?

This retrospective study was undertaken to investigate the observation that the probability of pregnancy was higher with intrauterine insemination (IUI) when human chorionic gonadotrophin (HCG) was administered after the onset of the luteinizing hormone (LH) surge. A total of 219 patients who had 524 IUI cycles was included in this study. IUI cycles were divided into three groups: group 1, patients who had an endogenous LH surge but no HCG; group 2, patients given HCG after an endogenous LH surge was observed; and group 3, patients given HCG before an endogenous LH surge could be demonstrated. The overall clinical pregnancy rate was 16%. Forty-two (19.2%) patients had 91 cycles with their partner's semen, while 177 (80.8%) used donor semen in 433 cycles; clinical pregnancy rates were 12.1% and 16.9% respectively. There was no significant difference in pregnancy rate per cycle between patients in group 1 (12.7%) compared with those in groups 2 (15.6%) or 3 (20.5%). We could not establish any benefit in waiting for a spontaneous LH surge before administering HCG in the presence of a mature follicle(s) in this study. This strategy avoids further monitoring to detect the LH surge, allowing treatment to be planned for a time convenient to the patient.

Postgraduate journal club as a means of promoting evidence-based obstetrics and gynaecology.

We assessed the feasibility of a new educational programme to teach critical appraisal of the medical literature to postgraduate trainees. The new programme used a journal club format where selection of topics and articles was driven by clinical problems arising in day-to-day practice. Papers were appraised critically according to validated guidelines using computer software for appraisal, electronic storage and retrieval. Over the initial 4-month period the journal club produced 17 critically appraised topics, two of which were published in peer-reviewed journals. During the study, trainees' reading time improved from a median of 2.0 hours (range 1-5 hours) to 3.5 hours (range 2-8 hours) (P = 0.026) and their knowledge scores improved from a mean of 50.8 (SD 4.0) to 62.9 (SD 4.3) (P = 0.003). We conclude that a journal club supported by electronic means of critical appraisal and dissemination of appraised information can be used to encourage the practice of evidence-based medicine.

Continuing the debate on empty follicle syndrome: can it be associated with normal bioavailability of beta-human chorionic gonadotrophin on the day of oocyte recovery?

This paper describes our experience with four ovarian stimulation in-vitro fertilization (IVF) cycles in which we failed to retrieve oocytes despite normal bioavailability of beta-human chorionic gonadotrophin (beta-HCG) in patients' blood 35 h after HCG administration. In three cases, the oocyte recovery procedure was interrupted, a second dose of HCG was administered and 24 h later mature oocytes were collected from two of the patients. In the first case, the three metaphase II oocytes collected fertilized after intracytoplasmic sperm injection (ICSI) and two cleaved grade three embryos were transferred but pregnancy did not ensue. In the second case, six out of eight metaphase II oocytes fertilized and cleaved following ICSI, leading to transfer of one grade two and two grade three embryos. This resulted in a clinical pregnancy which at the time of this report is ongoing. A similar rescue protocol was used for the third case who had empty follicle syndrome (EFS) in her previous treatment cycle but only cumulus-corona complexes were aspirated. Five additional patients who had EFS before instituting pregnancy diagnostic test screening have had further treatment cycles in which oocytes were collected but pregnancy did not ensue. We conclude that normal bioavailability of beta-HCG on the day of oocyte recovery does not exclude the diagnosis of EFS. EFS does not predict a reduced fertility potential in future cycles, although it may recur due to a biological abnormality in the availability of mature oocytes that are retrievable. In such patients, oocyte donation may offer the chance of achieving a pregnancy.

Contamination of embryo transfer catheter and treatment outcome in in vitro fertilization.

PURPOSE: The effect of embryo transfer (ET) catheter contamination with mucus and/or blood on treatment outcome in an in vitro fertilization (IVF) program was evaluated. METHODS: One thousand four ET procedures in 877 patients having conventional IVF in the long (1189 cycle) and short (15 cycles) protocol were analyzed to determine the impact of catheter contamination on the incidence of retained embryos and the clinical pregnancy rate. RESULTS: Catheter contamination with mucus and/or blood is a feature of difficult ET. Embryos were significantly more likely to be retained when the transfer catheter was contaminated with mucus (17.8 versus 3.3%) or blood (12 versus 3.3%). When the catheter was contaminated and an embryo(s) was retained and immediately retransferred, the pregnancy rate was not compromised. The pregnancy rate was significantly reduced when the ET catheter was contaminated with blood (15.5 versus 27.1%; P = 0.002), but no embryo was retained in the catheter set. CONCLUSIONS: Catheter contamination compromises the treatment outcome in IVF only when there is no associated retained embryo(s). As increased vigilance in searching for extruded embryos may not be practical, we suggest that cervical mucus should be routinely aspirated and ET performed as atraumatically as possible.

In vitro fertilization with low-dose clomiphene citrate stimulation in women who respond poorly to superovulation.

PURPOSE: Our experience with IVF using low-dose clomiphene citrate for stimulation in "non-" and "poor" responders was reviewed and the treatment outcomes with the previous controlled ovarian stimulation cycles in which hMG and GnRH agonist were used were compared. METHODS: The treatment outcome in 11 non- and 20 poor responders having 30 and 53 clomiphene citrate IVF treatment cycles, respectively, were compared with the treatment outcome in the previous long-protocol buserelin/hMG cycles. RESULTS: The clinical pregnancy rates per oocyte collection achieved in the first clomiphene citrate cycle in non (9.1%)- and poor (10%) responders were comparable to those achieved by poor responders (11.9%) who had buserelin/hMG using the long protocol. Although the numbers were small, a similar pregnancy rate could still be achieved in poor responders up to the third attempt using clomiphene citrate. CONCLUSIONS: IVF using long-protocol buserelin/hMG is more successful than using clomiphene citrate stimulation. However, this advantage may not be significant in those women with a previous poor response to buserelin/hMG. It is suggested that for such poor responders, three attempts of IVF in a clomiphene citrate cycle may offer a viable therapeutic alternative before reverting to more stressful, expensive, and time-consuming treatment.

Multiple attempts at embryo transfer: does this affect in-vitro fertilization treatment outcome?

In this study, we retrospectively analysed data from 877 patients who had 1204 embryo transfer procedures following in-vitro fertilization (IVF) at Midland Fertility Services, UK, between January 1991 and December 1995 to investigate the factors contributing to failure of embryo transfer at first attempt and the impact of immediate retransfer of retained embryos on the treatment outcome. Embryos were significantly more likely to be retained when the embryo transfer catheter was contaminated with mucus (3.3 versus 17.8%, P = 0.000001) or blood (3.3 versus 12%, P = 0.00001) and when the transfer procedure was difficult compared with when it was easy (20.3 versus 0.8%, P = 0.00001). There was no significant difference in the clinical pregnancy rate between those who had all their embryos transferred at the first attempt (24.7%) and those who required more than one attempt (23.2%). The types of embryo transfer catheter used in the unit did not show any difference in terms of embryo retention. Although we recommend aspiration of cervical mucus in order to reduce the rate of retained embryos, there is no evidence from our study to suggest that pregnancy rate is compromised when embryos are retained, provided they are discovered and immediately retransferred into the uterine cavity. Immediate retransfer is more convenient to the patients and reduces the laboratory workload without compromising the treatment outcome.

Elective cryopreservation of all embryos in women at risk of developing ovarian hyperstimulation syndrome may not prevent the condition but reduces the live birth rate.

OBJECTIVE: Our goal was to evaluate the use of elective cryopreservation of all embryos to prevent the development of ovarian hyperstimulation syndrome in patients at risk while undergoing in vitro fertilization treatment. DESIGN: We analyzed 117 treatment cycles in which the serum E2 concentration on the day of hCG administration was > 10,000 pM and in whom > or = 15 oocytes were retrieved at ultrasound-directed follicle aspiration. The incidence of ovarian hyperstimulation syndrome, pregnancy, and live birth in 65 patients who had elective cryopreservation of all embryos and 52 patients who had fresh embryo transfer were compared. Independent t test and chi-square test (with Yates' correction) was used as appropriate. RESULTS: The clinical pregnancy (35 vs 17%; P < 0.03) and the live birth (27 vs 12%; P < 0.05) rates in patients receiving fresh embryo transfer was significantly higher than in those who had elective cryopreservation of all embryos. The incidence of moderate and severe ovarian hyperstimulation syndrome was similar in both groups (3.8 and 6.2%). CONCLUSIONS: Elective cryopreservation of all embryos does not reliably protect against the development of ovarian hyperstimulation syndrome but may reduce the clinical pregnancy and live birth rate.

Outcome of frozen embryo replacement cycles following elective cryopreservation of all embryos in women at risk of developing ovarian hyperstimulation syndrome.

AIM: Our aim was to compare the outcome in subsequent frozen embryo replacement cycles in four groups of patients who had elective cryopreservation of all their embryos because they were considered to be at increased risk of developing severe ovarian hyperstimulation syndrome. DESIGN: Sixty-two (91%) of 68 IVF cycles (68 patients) in which elective cryopreservation of all embryos was performed were analyzed. All patients continued on the GnRH agonist, buserelin, after oocyte recovery until the onset of vaginal bleeding. Frozen embryo replacement occurred in a hormone replacement cycle that started either on day 3 of the withdrawal bleed (group I; N = 15) or after serum estradiol levels had fallen to < 100 pmol/L (group II; N = 16). The other patients commenced a frozen embryo replacement cycle several months later in either a hormone replacement (group III; N = 15) or a natural (group IV; N = 16) cycle. RESULTS: Two patients developed severe ovarian hyperstimulation syndrome. There were no significant differences among the four groups regarding demographic variables, the dose of hMG used, and the clinical outcome. There was a higher but not significantly different clinical pregnancy rate in group I (26.7%), compared to group II (12.5%), group III (13.3%), and group IV (18.8%). CONCLUSIONS: Several options exist for the timing and protocol used for frozen embryo replacement in patients who had elective cryopreservation for the prevention of ovarian hyperstimulation syndrome, none of which was found to be clearly superior in this observational report.

A prospective randomized study comparing needles of different diameters for transvaginal ultrasound-directed follicle aspiration.

OBJECTIVE: To determine whether the use of smaller diameter needles at oocyte collection reduces pain without jeopardizing the treatment outcome. DESIGN: Prospective randomized study. SETTING: The Assisted Conception Unit, Kings College Hospital, United Kingdom. PATIENTS: One hundred twelve patients undergoing an IVF-ET treatment cycle. INTERVENTIONS: Study patients were aged < or = 38 years and had at least six follicles > 14 mm on the day of oocyte collection. Patients were randomized to follicle aspiration with a 15- (n = 34), 17- (n = 41), or 18- (n = 37) gauge (g) single channel needle. Intravenous pethidine and diazepam were used for analgesia and sedation. Pain was quantified by means of questionnaires completed by the patient and by her partner postoperatively. RESULTS: A greater proportion of patients reported severe to unbearable pain with the 15-gauge compared with the 17-gauge (44% versus 22%) or 18-gauge (44% versus 16%) needles. Partners' perception of severe pain experienced by patients showed the same trend when the 15-gauge was compared with the 17- (62% versus 29%) and 18-gauge (62% versus 22%) needles. There was no significant difference between the needles regarding the oocyte collection rate, fertilization rate, proportion of oocytes with fractured zonae, cleavage rate, implantation rate, and pregnancy rate per cycle. CONCLUSIONS: Reducing the size of the needle used for oocyte collection from 15 to 17 or 18 gauge reduces pain without affecting the number of oocytes collected, their quality, or the clinical pregnancy rate.

Fetal macrosomia and pregnancy outcome in Lagos.

In an analysis of 6376 singleton births the prevalence of macrosomia was 4.9%; the attending perinatal mortality was 58/1000 compared to 18/1000 in controls. Eighty-three percent perinatal deaths occurred in unbooked patients after prolonged and neglected labor. Mortality and morbidity were weight related; the macrosomic baby delivered by section or by diabetic mother or that died was significantly heavier. There was strong association between maternal age, parity, diabetes, mild hypertension, previous history of a big baby and macrosomia in this study. Pregnancy was significantly prolonged with higher incidence of emergency sections and primary postpartum hemorrhage in mothers of macrosomic babies. Fetal sex does not appear to be an important factor in macrosomia.