Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle.

STUDY QUESTION: Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation? SUMMARY ANSWER: Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation. WHAT IS KNOWN ALREADY: Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows. STUDY DESIGN, SIZE, DURATION: Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbæk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Müllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested--besides LH--TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response. LIMITATIONS, REASONS FOR CAUTION: The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle. WIDER IMPLICATIONS OF THE FINDINGS: These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers. STUDY FUNDING/COMPETING INTEREST(S): Grants from the Research Fund of Region Sjælland are gratefully acknowledged. None of the authors declared any conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Impact of PCOS on early embryo cleavage kinetics.

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t2, t3, t4, t5, t6, t7, t8, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t2, t3, t4 and t7 but not at t5, t6, t8, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls.

GnRHa trigger and individualized luteal phase hCG support according to ovarian response to stimulation: two prospective randomized controlled multi-centre studies in IVF patients.

STUDY QUESTION: Does a GnRH agonist (GnRHa) trigger followed by a bolus of 1.500 IU hCG in a group of patients at risk of ovarian hyperstimulation syndrome (OHSS) reduce the OHSS incidence compared with hCG trigger? SUMMARY ANSWER: A GnRHa trigger followed by early luteal hCG support with one bolus of 1.500 IU hCG appears to reduce OHSS in patients at risk of OHSS; however, in a low-risk group a second bolus of 1.500 IU hCG induced two cases of late onset OHSS. WHAT IS KNOWN ALREADY: A GnRHa trigger is an alternative to hCG in GnRH antagonist co-treated cycles. STUDY DESIGN, SIZE, DURATION: Two RCTs were performed in four Danish IVF units. A total of 446 patients were assessed for eligibility and 390 patients were enrolled in the study from January 2009 until December 2011. The primary outcome of the study was OHSS incidence in the group at risk of OHSS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients received a fixed dose of recombinant human FSH for the first 4 days. On the day of triggering, patients were assessed for their risk of OHSS based on the total number of follicles ≥11 mm diameter, and were classified as being at risk of OHSS when the total number of follicles ≥11 mm was between 15 and 25 and at low risk of OHSS when the total number of follicles ≥11 mm was ≤14. Two separate randomization lists were used for each of the OHSS risk groups. Women at risk of OHSS were allocated (RCT 1) to either: Group A (n = 60), ovulation triggering with a bolus of 0.5 mg buserelin (GnRHa) s.c. followed by a single bolus of 1.500 IU hCG s.c. after the oocyte retrieval-or: Group B (n = 58): 5.000 IU hCG. Similarly, women at low risk of OHSS were allocated (RCT 2) to receive either: Group C (n = 125), a bolus of 0.5 mg buserelin s.c., followed by a bolus of 1.500 IU hCG s.c. after oocyte retrieval and a second bolus of 1.500 IU hCG on the day of oocyte retrieval +5-or: Group D (n = 141), 5.000 IU hCG. Groups C and D were included in order to obtain preliminary data. MAIN RESULTS AND THE ROLE OF CHANCE: In women at risk of OHSS (RCT 1) (15-25 follicles) no OHSS case was seen in Group A (GnRHa trigger and one bolus of 1.500 IU hCG), whereas two cases of moderate late-onset OHSS occurred in group B (3.4%), (P = 0.24). In contrast, in women at a low risk of OHSS (RCT 2) (≤14 follicles) two cases of late-onset OHSS occurred in Group C (GnRHa trigger and two boluses of 1.500 IU hCG), whereas no OHSS case was encountered in Group D (P = 0.22). LIMITATIONS, REASONS FOR CAUTION: Although the first RCT was powered to include 168 patients at risk of OHSS (15-25 follicles ≥11 mm) randomized to either GnRHa trigger or hCG trigger, the trial was prematurely discontinued when a total of 118 patients at risk of OHSS were randomized. In addition the second RCT in the OHSS low-risk group was designed as a feasibility study to assess the incidence of OHSS after GnRHa trigger and dual hCG administration versus 5.000 IU hCG. No power calculation was performed for this trial. In addition, there was a lack of blinding in the RCTs. WIDER IMPLICATIONS OF THE FINDINGS: Although a non-significant result, one bolus of 1.500 IU hCG after GnRHa trigger tended to reduce the OHSS rate in patients with 15-25 follicles ≥11 mm as well as secure the ongoing pregnancy rate. In contrast, in patients at low risk of OHSS the administration of two boluses of 1.500 IU hCG after GnRHa trigger should be avoided as it may induce OHSS.

The impact of pronuclei morphology and dynamicity on live birth outcome after time-lapse culture.

STUDY QUESTION: Can the pronuclei (PN) morphology and the time of PN breakdown (PNB) predict the potential of embryos to result in live birth? SUMMARY ANSWER: In comparison to embryos resulting in no live birth, PNB occurred significantly later in embryos resulting in live birth and never earlier than 20 h 45 min. None of the tested scoring systems were shown to predict the live birth outcome in a time-lapse set-up. WHAT IS KNOWN ALREADY: The PN morphology is supported as a prominent embryo selection parameter in single light microscopy observations, although controversial results have been reported. STUDY DESIGN, SIZE, DURATION: This was a prospective study of 159 embryos, all of which were later transferred. The PN morphology of 46 embryos which resulted in live birth was compared with that of 113 embryos which resulted in no live birth. PARTICIPANTS, SETTING: From 1 March 2010 to 30 August 2011, 130 couples underwent fertility treatment by ICSI. Embryo culture was performed in a time-lapse set-up from fertilization to intrauterine transfer. PN morphological assessment was performed on every embryo replaced, using six different scoring systems at different times. MAIN RESULTS AND THE ROLE OF CHANCE: No embryo with PNB earlier than 20 h 45 min resulted in live birth. All six PN assessment models showed no significant distribution of scores (P = NS) between the live birth and no live birth groups at 16 h post-fertilization (PF), 18 h PF and 40 min before PNB. The outcomes of assessments changed significantly (P < 0.001) over time and the time of PNB was found to be the optimal stage to evaluate the PN morphology. LIMITATIONS, REASONS FOR CAUTION: The study includes only embryos reaching the 4-cell stage after ICSI, and transferred at 44 h PF. WIDER IMPLICATIONS OF THE FINDINGS: The PN morphology changes over time, indicating that the single light microscopy observation approach is deficient in comparison to time-lapse. Although the assessment of the PN morphology does not improve embryo selection, the timing of PNB should be included in embryo selection parameters.

Polycystic ovary syndrome: infertility, cardiovascular, metabolic and obstetrical risks, laboratory and clinical outcomes -- the PICOLO Study.

OBJECTIVES: The primary objective of this multicenter study is to evaluate the relative impact of insulin resistance (IR) and body mass index (BMI) in women with polycystic ovary syndrome (PCOS) on (1) Key hemodynamic/thrombogenic variables, (2) Oocyte quality and early embryo development, (3) Fetal growth, placental function and adverse obstetric outcome. SECONDARY OBJECTIVE: To establish a PCOS database and biobank facilitating future basic and interventional research related to PCOS. DESIGN: A cross-sectional and longitudinal cohort study at four University Hospitals in Denmark. POPULATION INCLUSION: About 200 women fulfilling the Rotterdam Criteria and 100 women without PCOS recruited from 2010 to 2012. METHODS: The impact of PCOS, as well as the impact of IR and BMI on the hormonal, metabolic and hemostatic key variables will be analyzed combining conventional, molecular techniques and selected gene analysis. Oocytes will be characterized by gene expression of granulosa and cumulus cells and the early embryo development will be followed by time lapse microscopy. Fetal growth will be assessed by repeated ultrasound measurements, and the pregnancy outcome compared to maternal and fetal biochemical markers of growth and inflammation and clinical pregnancy complications. MAIN OUTCOME MEASURES: Metabolic and hemostatic risk-biomarkers, oocyte and embryo quality, adverse pregnancy outcome, fetal growth and placental function in women with PCOS.

Influence of the dominant follicle on in-vitro maturation of human oocytes: a prospective non-randomized study.

The present study was designed to examine the influence of timing of aspiration and the influence of a dominant follicle on maturation and fertility potential of immature oocytes aspirated in unstimulated cycles. The study included 81 regularly cycling women. In group I (n = 53), oocyte retrieval was scheduled the day after a follicle of 10 mm and an endometrium of at least 5 mm were observed. In group II (n = 28), aspiration was scheduled the day after observation of the same ultrasound criteria plus a detected increase (100%) in the level of oestradiol compared with the level on day 3. The maturation rate was significantly higher in group I compared with group II (107/184, 58.2% versus 56/124, 45.2%, P < 0.05), whereas the rates of fertilization and cleavage did not differ between the two groups. The clinical pregnancy rate per aspiration was significantly higher in group I compared with group II (9/53, 17% versus 0/28, 0%, P < 0.05). When comparing oocytes originating from the ovary with the dominant follicle (ipsilateral ovary) with oocytes originating from the ovary without a dominant follicle (contralateral ovary) an increased fertilization rate was observed in group I, and an increased maturation rate was observed in group II. When the data from the two groups were pooled, an increased maturation rate was observed in oocytes originating from the ipsilateral ovary compared with oocytes originating from the contralateral ovary. No difference was found with respect to rates of fertilization and cleavage rates when all oocytes originating from the ipsilateral ovary were compared with all oocytes originating from the contralateral ovary.

Maternal serum supplementation in culture medium benefits maturation of immature human oocytes.

This study compared the rates of maturation, fertilization, cleavage and pregnancy among oocytes matured in medium containing either human serum albumin (HSA) or maternal serum. Immature oocytes were obtained from 51 consecutive regularly cycling women <38 years of age. Immature oocytes were aspirated transvaginally on cycle day 8-9 after priming with FSH (Gonal-F 150 IU/day for 3 days, initiated on day 3). Oocytes were matured in Dyrkningsmedie til IVM supplemented with recombinant FSH (rFSH) 0.075 IU/ml and HCG 0.5 IU/ml for 28-30 h. In group I (n = 63 oocytes obtained from the first 23 cycles) the culture medium was supplemented with 2% (w/v) HSA. In group II (n = 74 oocytes obtained from the following 28 cycles) the medium was supplemented with 10% (v/v) heat-inactivated maternal serum. Intracytoplasmic sperm injection (ICSI) was performed on all methaphase II oocytes. Significantly increased rates of maturation 47/74 (63%) vs. 26/63 (41%) (P < 0.05), pregnancy 6/28 (21%) vs. 0/23 (0%) (P < 0.05) and implantation 6/20 (30%) vs. 0/15 (0%) (P < 0.05) were obtained from oocytes matured in culture medium with maternal serum supplementation compared with oocytes matured in medium supplemented with HSA. These results indicate that factors other than albumin in maternal serum play an important role in maturation and subsequent developmental capacity of human oocytes.

Preoperative treatment with oestradiol in women scheduled for vaginal operation for genital prolapse. A randomised, double-blind trial.

OBJECTIVE: To disclose a clinical and histopathological effect of local low-dose oestradiol treatment on the vagina. DESIGN: A randomised, double-blind trial. SETTING: Two gynaecological departments at University Hospitals. SUBJECTS: Forty-eight postmenopausal women scheduled for surgery because of genital prolapse. INTERVENTION: 25 micrograms oestradiol or placebo, administered as vaginal pessaries daily, 3 weeks prior to surgery. MAIN OUTCOME MEASURES: Cytological, histological and clinical changes of the vaginal mucosa. RESULTS: The thickness of the vaginal wall increased as did the oestrogenic index. No clinical effect was seen apart from decreased incidence of recurrent cystitis postoperatively. CONCLUSIONS: Preoperative oestrogen treatment has been shown to reduce the incidence of recurrent cystitis and may be needed for stimulation of vaginal mucosa; the short-term clinical effect is not convincing, however.

The effect of hydroxyprogesterone on ureteral stones.

It has been suggested that dilatation of the ureter seen in pregnancy is caused by progesterone. Twenty-four patients with ureteral calculi, therefore, were given an intramuscular injection of 250 mg hydroxyprogesterone in order to facilitate the discharge of the stones. Irrespective of the duration of symptoms there was a potential effect of treatment in up to 66% of the patients. In patients with symptoms of less than three weeks duration the stones were discharged within three weeks after the first symptom in 75% of the cases, which is a much higher incidence rate than that stated in the literature for spontaneous stone discharge (18-30%). In patients with longstanding symptoms 50% of the stones had passed, as compared to 39% reported in the literature. Especially concrements with a diameter larger than 4 mm had a better discharge rate. As the treatment is simple, cheap and without side effects, it seems worthwhile trying it in order to avoid surgery.

The value of peroperative ultrasound examination in first trimester legally induced abortion.

A prospective study was carried out based on 117 first trimester legally induced abortions. Abdominal ultrasound examination was performed immediately after aspiration. The predictive value of an empty uterus at ultrasound examination was 97.3%. The rate of retained tissue was not reduced compared to studies where peroperative ultrasound was not used.

PIP: Despite curettage after vacuum aspiration of induced first trimester abortions, some patients experience signs and symptoms of retained placental tissue. A prospective study was conducted to evaluate the use of postoperative ultrasound examination in the prediction of an empty uterus in induced abortion and to study the effect of preoperative ultrasound on the rate of complications after legal termination in the first trimester. The study was conducted among 117 women, 18-48 years old, who were referred for legally induced first trimester abortion with their consent. The abortions were performed under general anaesthesia by vacuum aspiration followed by blunt curettage of the cavity, by surgeons with varying experience. 10 iu of oxytocin were given intravenously to each women at the end of the operation. Abdominal ultrasound examination was performed after the completion of the procedure. The uterus was examined longitudinally and transversely using an Ultrasound Scanner type 1846 equipped with a 3 mhz abdominal transducer. Dense echoes in the uterus cavity indicated retained tissue and the procedure with aspiration and/or curettage was performed repeatedly until a clearly defined echo cavity was found. The women were discharged the same day. Complications requiring readmission within the first month were registered. No preoperative complications were registered. 6 women were readmitted within one month after abortion because of bleeding and pain, and recurettage was performed in 5 women because of clinical signs of retained tissue. Retained pregnancy products were demonstrated histopathologically only in 3 patients, while another 2 patients were treated with antibiotics because of asymptomatic positive chlamydia culture. The study demonstrated a predictive value of an empty uterus of 97.3% at ultrasound examination. The rate of retained tissue was not reduced compared to other studies that did not use ultrasound examination. The findings of the study therefore suggest that ultrasound examination is valuable when retention of products is suspected, but it cannot be recommended as routine for all patients.

Changes in plasmaconcentration of ANP and aldosterone in normal pregnancy and pregnancy complicated by pre-eclampsia.

Changes in plasmaconcentration of ANP in normal pregnancy and pregnancy complicated by pre-eclampsia were examined and compared to non-pregnant controls. The maternal plasma concentration increased gradually during normal pregnancy but values did not deviate significantly from non-pregnant women. A further increase was demonstrated post partum. In pre-eclampsia maternal plasma ANP levels increased significantly compared with normal gravida at the same gestational age and non-pregnant controls. After delivery ANP decreased significantly. Non correlation between ANP and aldosterone were found in either normal pregnancy nor pregnancy complicated by pre-eclampsia. It is not yet clear how important ANP is for the regulation of blood pressure and sodium and water balance during normal pregnancy and pregnancy complicated by pre-eclampsia.

[Severe male infertility. Genetic investigation and counseling prior to intracytoplasmic sperm injections]. / Mandlig infertilitet i svaer grad. Genetisk udredning og rådgivning før intracytoplasmatisk saedcelleinjektion.

Intracytoplasmatic sperm injection (ICSI) has improved the success rate in treating severe male infertility. The method may now be used with sperm from the epididymis and testis. This article summarizes our knowledge on genetic factors affecting male gamete formation or function. Infertile men with severe impairment of spermatogenesis showed a higher than normal incidence of chromosomal abnormalities and 10-20% had microdeletion, in the Y-chromosome. About 75% of males with congenital bilateral absence of vas deferens (CBAVD) have mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene. In conclusion, we recommend genetic counselling to all couples with a diagnosis of male infertility prior to ICSI. Men with severe oligozoospermia or non-obstructive azoospermia should have karyotype analysis performed and with establishment of diagnostic tools to reveal Y-chromosome deletions, this should be offered to the same group of men. Men with obstructive azoospermia and congenital albilateral absence of vas deferens as well as their wives should be screened for cystic fibrosis mutations.

Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study.

The aim of this study was to determine whether the rates of in vitro oocyte maturation, fertilization and cleavage, as well as implantation rate and pregnancy rate, could be improved by low-dose priming with FSH in vivo before retrieval of immature oocytes in patients with polycystic ovary syndrome (PCOS). From March 1998 to June 2000, a total of 28 women underwent 36 completed treatment cycles, randomized sequentially in one of two groups. Women in group 1 (n = 12 cycles) received no stimulation and women in group 2 (n = 24 cycles) received 150 iu recombinant FSH day(-1) for 3 days, initiated on day 3 after menstruation. Aspiration was performed transvaginally between day 9 and day 17 in the unstimulated group and on day 8 or day 9 in the FSH-primed group after FSH deprivation for 2 or 3 days. All cumulus-enclosed oocytes of healthy appearance were matured in culture medium (TCM-199) in vitro for 28-36 h before intracytoplasmic sperm injection (ICSI). After oocyte retrieval the women were given oestradiol (6 mg day(-1)) and progesterone administration (300 mg day(-1)) was initiated 2 days later. Suitable embryos (maximum two embryos) were transferred on day 3 after ICSI. The percentage of oocytes reaching metaphase II was significantly higher (P < 0.05) in the FSH-primed group (59%, 92/156) compared with the non-primed group (44%, 36/81). There were no significant differences in the rates of oocyte fertilization and cleavage between these groups. No pregnancies were obtained in group 1 (0%, 0/12), whereas seven clinical pregnancies were obtained in group 2 (29%, 7/24) (P < 0.05). In group 2, 37 embryo transfers resulted in eight implantations (21.6%). Three healthy singleton children have been born at term; the remaining pregnancies ended with spontaneous abortions in the first trimester. These results indicate that priming with recombinant FSH before harvesting of immature oocytes from patients with PCOS may improve the maturational potential of the oocytes and the implantation rate of the cleaved embryos.

Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality.

BACKGROUND: The purpose of the present study was to investigate the morphology of in-vitro matured metaphase II (MII) oocytes and to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of in-vitro matured MII oocytes was related to their subsequent fertilization and cleavage rates and to embryo quality. METHODS: This retrospective study included 264 MII oocytes obtained in 100 consecutive cycles. Oocyte retrieval was performed transvaginally and cumulus enclosed oocytes were matured for 28--30 h before evaluation. Prior to ICSI, all MII oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly (n = 144, 54%), grade II: oocytes with one anomaly (n = 87, 33%) and grade III: oocytes with at least two anomalies (n = 33, 12.5%). RESULTS: Oocyte grades did not differ between women with polycystic ovaries [grades I, II and III respectively: 58/94 (61.7%), 29/94 (30.9%) and 7/94 (7.4%)] and women with normal ovaries [grades I, II and III respectively: 86/170 (50.6%); 58/170 (34%); 26/170 (15.3%)]. Morphology was not related to fertilization rates. The cleavage rate was, however, affected by morphological anomalies (grade I [77/144 (53.5%) versus grade II 33/87 (37.9%) (P = 0.03)], although no significant decrease in cleavage rate could be demonstrated when all grade II and III oocytes were compared with normal oocytes. Significantly more embryos of good quality developed after grade I oocytes [54/144 (37.5%)] compared with those from grade II and grade III oocytes (22/120; P = 0.001). The presence of cytoplasmic abnormalities significantly decreased the cleavage rate (P = 0.04) and also the number of good quality embryos (P < 0.001). CONCLUSION: The in-vitro maturation of oocytes without anomalies yields higher quality embryos, with higher cleavage rates, than those with anomalies.

Laparoscopy and ultrasound examination in women with acute pelvic pain.

The results of preoperative pelvic examination and eventual ultrasound examination were correlated with the laparoscopic findings in 316 women with acute pelvic pain. The predictive values of normal and abnormal findings at pelvic examination were 46.9 and 82.1%, respectively. 42.1% of the women had ultrasound examination performed. This investigation showed to be helpful especially in patients with normal findings at pelvic examination. If ultrasonic findings were abnormal the results at laparoscopy were also abnormal in 90%. On the contrary, normal findings at ultrasound examination did not exclude abnormal pelvic findings. The predictive value of normal results at ultrasound examination was 50.0%. This discrepancy between ultrasonic and pelvic findings can be explained by the size of the pelvic masses. Ultrasound examination is a valuable tool in the evaluation of patients with acute pelvic pain, but it cannot replace laparoscopy.

Leiomyoma of the urinary bladder.

We report a case of urinary retention in a women caused by a leiomyoma of the bladder. The intramural form often causes obstruction, as it is usually located near the bladder base. Treatment is surgical, and the prognosis is excellent. Therefore, cystoscopy is recommended in women with urinary retention.

Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation.

The purpose of the present study was to analyse the results of maturation of oocytes obtained in unstimulated normal women after a leading follicle of 10 mm diameter and an endometrium of at least 5 mm thickness were observed at ultrasound. The serum concentrations of oestradiol and inhibin A were analysed from day 3 to the day of aspiration and retrospectively evaluated. A total of 75 normal regular cycling women referred for IVF/intracytoplasmic sperm injection (ICSI) because of male factor and/or tubal disease was included (n = 87 consecutive cycles). The oocytes were aspirated transvaginally and matured for 28-36 h. ICSI was performed on all metaphase II oocytes and they were cultured to day 2 or 3 after insemination, at which time suitable embryos (maximum two) were replaced into the women. Eleven singleton pregnancies with a live fetus were obtained after transfer in 63 cycles (pregnancy rate of 13% per aspiration and 17% per transfer). Nine healthy children have been born and the remaining two pregnancies miscarried in the eighth to ninth gestational week. The serum concentrations of oestradiol on day 3 and on the day of aspiration were available in 85 cycles, and in 57 of these an increase of 100% was detected on the day of aspiration. Significantly more pregnancies were observed in these cycles compared with cycles without an increase in the concentration of oestradiol (19 versus 0% per aspiration, 24 versus 0% per transfer, P < 0.02). Further improvement in pregnancy rate was obtained if aspiration was performed after increase in inhibin A concentration (24 versus 0% per aspiration and 33 versus 0% per transfer, P < 0.02) (n = 83 cycles, where inhibin concentrations were available, and n = 42 cycles, where an increase of 80% was detected on the day of aspiration compared with day 3). Timing of aspiration may improve the developmental potential of immature oocytes.

Possible factors affecting the development of oocytes in in-vitro maturation.

To date, pregnancy rates from oocytes matured in-vitro (IVM) have been much lower than those with in-vivo stimulated maturation. In order to improve the developmental potential of IVM oocytes, we studied the effect of three possible factors on pregnancy rates: (i) priming in vivo with FSH before aspiration; (ii) the time interval of maturation in vitro, and (iii) timing the aspiration by monitoring the serum concentrations of oestradiol and inhibin A. In all experiments, oocyte retrieval was performed transvaginally and oocytes were matured individually in culture medium (TCM 199) under oil. Intracytoplasmic sperm injection (ICSI) was carried out on all metaphase II oocytes. Suitable embryos (maximum of two) were replaced after culturing for 2-3 days in IVF medium. Endometrial priming consisted of 2 mg 17beta-oestradiol, taken orally three times a day from the day of oocyte retrieval, and intravaginal progesterone suppositories initiated 2 days later. In the first experiment, 20 women were randomly allocated to two groups: group I (n = 10 cycles) received no stimulation, while group II (n = 10 cycles) received recombinant FSH 150 IU/day for 3 days, initiated on day 3. FSH priming did not affect the rates of maturation, fertilization or cleavage, and no effect was seen on embryo development. The second experiment included 48 patients undergoing 55 unstimulated cycles. The effects of IVM periods of 28 and 36 h were compared. Shortening the IVM period did not compromise subsequent embryo development. The third study analysed the results of maturation of oocytes obtained in 87 cycles in 75 unstimulated normal women, after a leading follicle of 10 mm in diameter and an endometrial thickness of at least 5 mm were observed. A pregnancy rate of 12.6% (11/87) per aspiration and 17.4% (11/63) per transfer was obtained. Serum concentrations of oestradiol and inhibin A were evaluated retrospectively. Significantly more pregnancies were obtained in cycles with a detected increase in the concentration of oestradiol from day 3 to the day of aspiration (19% per aspiration) compared with cycles without such an increase (0% per aspiration). A higher pregnancy rate was observed after an increase in inhibin A concentration (24 versus 0%). In conclusion, monitoring the timing of aspiration may potentially improve the developmental potential of immature oocytes. FSH priming did not affect either cleavage rate or embryo development. Shortening the maturation period from 36 to 28 h did not compromise subsequent embryonic development.