Anne Lis Mikkelsen

Strategies in human in-vitro maturation and their clinical outcome.

The basis of in-vitro maturation (IVM) is the maturing in vitro of oocytes from the germinal vesicle (GV) stage of development to the metaphase II stage. Experience in handling immature oocytes has been obtained from two main groups. The first group is women suffering from polycystic ovarian syndrome, who are extremely sensitive to stimulation with exogenous gonadotrophins in assisted reproduction, and have a significant risk of developing ovarian hyperstimulation syndrome (OHSS). The second group is regular cycling women with normal ovaries referred for IVF due to severe male infertility. In both groups, aspiration of immature oocytes has been performed in unstimulated cycles and after priming with human chorionic gonadotrophin or FSH respectively. Clinical pregnancy rates of 24% per aspiration have been obtained. Children born after IVM appear to be healthy. These data, taken together, suggest that in future, immature oocyte retrieval combined with IVM could replace conventional IVF in selected patients.

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.

Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the corresponding oocyte

Background. The purpose of this study was to determine the incidence of apoptosis in cumulus cells, and correlate these findings with the maturation stage, fertilization rate and embryo score of the corresponding oocyte, in couples undergoing ICSI due to a male factor.

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.

Differential expression of inflammation-related genes in the ovarian stroma and granulosa cells of PCOS women

Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder. Ovarian changes in PCOS women are well characterized by ultrasound. However, the ovarian pathophysiology is not fully understood. The aim of this study was to characterize the expression, in both the central ovarian stroma and in granulosa cells (GCs), of a number of genes, including several inflammation-related genes, which have been hypothesized to be involved in the pathophysiology of PCOS. Biopsies of the central ovarian stroma were obtained from PCOS women (Rotterdam criteria) and from normally ovulating women in follicular phase. GCs were retrieved from PCOS-women and non-PCOS women, undergoing in vitro maturation. The expressions of 57 genes were analyzed by quantitative-PCR using a low-density-gene array. The main outcome measures were over-expression or under-expression of the specific genes. The results showed that in the central stroma of PCOS ovaries, five inflammation-related genes (CCL2, IL1R1, IL8, NOS2, TIMP1), the leukocyte marker CD45, the inflammation-related transcription factor RUNX2 and the growth factor AREG were under-expressed. The growth factor DUSP12 and the coagulation factor TFPI2 were over-expressed. In the GC of PCOS, all of the differentially expressed genes were over-expressed; the inflammation-related IL1B, IL8, LIF, NOS2 and PTGS2, the coagulation-related F3 and THBS1, the growth factors BMP6 and DUSP12, the permeability-related AQ3 and the growth-arrest-related GADD45A. In conclusion, the results indicate major alterations in the local ovarian immune system of PCOS ovaries. This may have implications for the PCOS-related defects in the inflammation-like ovulatory process and for the susceptibility to acquire the inflammatory state of ovarian hyperstimulation syndrome.

Levels of the epidermal growth factor-like peptide amphiregulin in follicular fluid reflect the mode of triggering ovulation: a comparison between gonadotrophin-releasing hormone agonist and urinary human chorionic gonadotrophin

OBJECTIVE To detect differences in follicular fluid (FF) levels of amphiregulin (AR), depending on mode of triggering final oocyte maturation. DESIGN Prospective randomized trial. SETTING Three IVF units. PATIENT(S) Ninety-six patients undergoing IVF-intracytoplasmic sperm injection. INTERVENTION(S) Ovulation triggered with either urinary hCG or GnRH agonist (GnRH-a). CONTROLS 15 FF samples from small antral follicles (3-9 mm) and 12 FF samples from natural cycle. MAIN OUTCOME MEASURE(S) Follicular fluid concentration of AR, P4, E2, vascular endothelial growth factor, and inhibin B. RESULT(S) Significantly lower levels of AR were found in FF from the GnRH-a group versus the hCG group, 51±3.5 versus 71±6.0 ng/mL. In FF from natural cycles, levels of AR were significantly higher than those of GnRH-a triggering but significantly lower than those of urinary hCG triggering. In small antral follicles only 5 out of 15 follicles contained measurable amounts of AR. When urinary hCG and GnRH-a triggering were compared, FF P4 was significantly higher after urinary hCG triggering, whereas no difference was seen regarding E2, vascular endothelial growth factor, and inhibin B. A total of 14% more metaphase II oocytes and 11% more transferable embryos were obtained after GnRH-a triggering. CONCLUSION(S) This study suggests that oocyte competence is linked to granulosa cell AR secretion.

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.

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.

PLASMA CONCENTRATION OF ATRIAL NATRIURETIC PEPTIDE IN NORMAL PREGNANT WOMEN AND IN PREGNANT WOMEN WITH PREECLAMPSIA

Plasma concentration of atrial natriuretic peptide (ANP) was determined in pregnant women with preeclampsia, in normal pregnant and in nonpregnant women by a specific radioimmunoassay. Results did not show important differences between nonpregnant controls and normal pregnant women, but a significant rise was seen in women with preeclampsia compared to nonpregnant controls. Marked interindividual variation was found in all three groups. The mechanism of ANP release may differ between those women with normal pregnancy and those with preeclampsia. It is unclear whether the increased level of ANP in preeclampsia is an effect or a cause of the disease.

Time interval between FSH priming and aspiration of immature human oocytes for in-vitro maturation: a prospective randomized study

This prospective randomized controlled study was performed to examine the influence of coasting for 2 days versus 3 days following a fixed daily dose of FSH for 3 days. The outcome was 2-fold. In the first experiment (n = 50 cycles), the incidence of apoptosis in granulosa cells was compared. In the second experiment (n = 28 cycles), the rates of maturation, fertilization, cleavage, pregnancy and implantation were compared. In addition, clinical pregnancy rate per aspiration was registered. Granulosa cells were collected from follicular aspirates and pooled for each patient. The APOPTAG Detection Kit was used for staining of the granulosa cells and detection of apoptosis. Oocytes were matured in vitro for 28-30 h before intracytoplasmic sperm injection. The incidence of apoptosis in granulosa cells did not differ between granulosa cells obtained after 2 days coasting (n = 25 cycles) compared with granulosa cells obtained after 3 days coasting (n = 25 cycles) (26.2 versus 26.2%). When oocytes obtained after coasting for 2 days (n = 12 cycles) were compared with oocytes obtained after coasting for 3 days (n = 16 cycles), no significant difference was found between rates of maturation (63 versus 65%), fertilization (60 versus 68%), cleavage (86 versus 92%) or implantation [5/12; 42 versus 1/12 (8%)]. A higher clinical pregnancy rate per aspiration [5/16 (31%) versus 1/12 (8%)] was obtained after coasting for 3 days compared with coasting for 2 days. The difference was not significant. This randomized study showed no difference in apoptosis of granulosa cells and no difference in developmental competence of oocytes obtained after coasting for 3 days compared with 2 days coasting.