No major differences in perinatal and maternal outcomes between uninterrupted embryo culture in time-lapse system and conventional embryo culture.

STUDY QUESTION: Is embryo culture in a closed time-lapse system associated with any differences in perinatal and maternal outcomes in comparison to conventional culture and spontaneous conception? SUMMARY ANSWER: There were no significant differences between time-lapse and conventional embryo culture in preterm birth (PTB, <37 weeks), low birth weight (LBW, >2500 g) and hypertensive disorders of pregnancy for singleton deliveries, the primary outcomes of this study. WHAT IS KNOWN ALREADY: Evidence from prospective trials evaluating the safety of time-lapse incubation for clinical use show similar embryo development rates, implantation rates, and ongoing pregnancy and live birth rates when compared to conventional incubation. Few studies have investigated if uninterrupted culture can alter risks of adverse perinatal outcomes presently associated with IVF when compared to conventional culture and spontaneous conceptions. STUDY DESIGN, SIZE, DURATION: This study is a Swedish population-based retrospective registry study, including 7379 singleton deliveries after fresh embryo transfer between 2013 and 2018 from selected IVF clinics. Perinatal outcomes of singletons born from time-lapse-cultured embryos were compared to singletons from embryos cultured in conventional incubators and 71 300 singletons from spontaneous conceptions. Main perinatal outcomes included PTB and LBW. Main maternal outcomes included hypertensive disorders of pregnancy (pregnancy hypertension and preeclampsia). PARTICIPANTS/MATERIALS, SETTING, METHODS: From nine IVF clinics, 2683 singletons born after fresh embryo transfer in a time-lapse system were compared to 4696 singletons born after culture in a conventional incubator and 71 300 singletons born after spontaneous conception matched for year of birth, parity, and maternal age. Patient and treatment characteristics from IVF deliveries were cross-linked with the Swedish Medical Birth Register, Register of Birth Defects, National Patient Register and Statistics Sweden. Children born after sperm and oocyte donation cycles and after Preimplantation Genetic testing cycles were excluded. Odds ratio (OR) and adjusted OR were calculated, adjusting for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: In the adjusted analyses, no significant differences were found for risk of PTB (adjusted OR 1.11, 95% CI 0.87-1.41) and LBW (adjusted OR 0.86, 95% CI 0.66-1.14) or hypertensive disorders of pregnancy; preeclampsia and hypertension (adjusted OR 0.99, 95% CI 0.67-1.45 and adjusted OR 0.98, 95% CI 0.62-1.53, respectively) between time-lapse and conventional incubation systems. A significantly increased risk of PTB (adjusted OR 1.31, 95% CI 1.08-1.60) and LBW (adjusted OR 1.36, 95% CI 1.08-1.72) was found for singletons born after time-lapse incubation compared to singletons born after spontaneous conceptions. In addition, a lower risk for pregnancy hypertension (adjusted OR 0.72 95% CI 0.53-0.99) but no significant difference for preeclampsia (adjusted OR 0.87, 95% CI 0.68-1.12) was found compared to spontaneous conceptions. Subgroup analyses showed that some risks were related to the day of embryo transfer, with more adverse outcomes after blastocyst transfer in comparison to cleavage stage transfer. LIMITATIONS, REASONS FOR CAUTION: This study is retrospective in design and different clinical strategies may have been used to select specific patient groups for time-lapse versus conventional incubation. The number of patients is limited and larger datasets are required to obtain more precise estimates and adjust for possible effect of additional embryo culture variables. WIDER IMPLICATIONS OF THE FINDINGS: Embryo culture in time-lapse systems is not associated with major differences in perinatal and maternal outcomes, compared to conventional embryo culture, suggesting that this technology is an acceptable alternative for embryo incubation. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by a research grant from Gedeon Richter. There are no conflicts of interest for all authors to declare. TRIAL REGISTRATION NUMBER: N/A.

No benefit of culturing embryos in a closed system compared with a conventional incubator in terms of number of good quality embryos: results from an RCT.

STUDY QUESTION: Does culture in a closed system result in an increased number of good quality embryos (GQE) on Day 2 compared with culture in a conventional system? SUMMARY ANSWER: Culture in a closed system up to 2 days after microinjection results in similar embryo development and morphological quality compared with culture in a conventional incubation system. WHAT IS KNOWN ALREADY: Time-lapse imaging (TLI) incubators are rapidly being introduced into IVF laboratories worldwide, despite the lack of large prospective randomized trials demonstrating improvement in embryo development or pregnancy rates. STUDY DESIGN, SIZE, DURATION: A randomized controlled trial including 364 patients (365 cycles) was conducted between May 2010 and February 2014. After oocyte collection, randomization was carried out and all of a patients' oocytes were allocated to culture in either a conventional incubator or a closed incubator system in proportion 1:2 until embryo transfer on Day 2. A total of 1979 oocytes were injected and cultured in the closed system, and 1000 in the standard incubator. The primary end-point was the number of GQE in the two groups. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: In total, 364 patients undergoing their first IVF cycle using ICSI, where at least one oocyte was retrieved, were randomized in a university hospital setting. Two hundred and forty patients were randomized for culture in a closed system and 124 patients for culture in the conventional incubator system (control group). Embryo assessments and final morphological scoring before transfer and cryopreservation were carried out at the same time points for embryos cultured in the conventional incubator and in the closed system. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the mean ± SD number of GQEs between groups: 2.41 ± 2.27 for the closed system group and 2.19 ± 1.82 for the control group (P = 0.34, difference 0.23, 95% confidence interval 0.69; -0.24). No significant differences were found in the number of 4-cell embryos, implantation-, pregnancy- or ongoing pregnancy rates. A significantly higher miscarriage rate was found in the TLI group compared with the control group (33.3 and 10.2%, P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Culture media, temperature and gas levels were similar in the open and closed incubator systems, but different culture dishes were used. Culturing embryos for longer time period (to the blastocyst stage) may give different results. Only ICSI patients were included, which may limit the generalizability of the results. Finally, the number of GQEs on Day 2 was used as a surrogate outcome for live birth. WIDER IMPLICATIONS OF THE FINDINGS: The results are consistent with other, smaller randomized trials showing no difference in embryo quality when comparing culture in a conventional incubator with that of a closed TLI incubator system.

Re-analysis of 166 embryos not transferred after PGS with advanced reproductive maternal age as indication.

BACKGROUND: In a randomized controlled study aiming to test the effectiveness of preimplantation genetic screening (PGS) in women of advanced maternal age, embryos diagnosed as chromosomally abnormal and those with no diagnosis were fixed for reanalysis. The aim of this study was to determine how well the chromosomal constitution of one biopsied blastomere reflects the status of the entire embryo. METHODS: One hundred and seventy-three embryos diagnosed as chromosomally abnormal, 22 with no PGS result and four degenerated embryos originally diagnosed as normal were fixed and reanalysed by fluorescence in situ hybridization. RESULTS: In total, 199 embryos were fixed, of which 166 were successfully reanalysed. One hundred and sixty embryos were found to be chromosomally abnormal; 48 of the reanalysed embryos with an initial diagnosis (149) had at least one cell with exactly the same chromosomal constitution shown in the first PGS analysis (34.2%). The reanalysis confirmed the initial overall chromosomally abnormal status of the embryo in 95.9% of the cases. Of all chromosomally abnormal embryos, 4.1% were diagnosed as false positive. The risk for false negative rate was at least 4.1%. CONCLUSIONS: PGS seems to be a good method for selecting against chromosomally abnormal embryos but not for determining an embryo's exact chromosomal constitution.

FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology.

BACKGROUND: The data are compiled from two multicentre, prospectively randomized studies on the effect of follicular fluid meiosis-activating sterol (FF-MAS) on human oocytes. The donated oocytes were exposed either to test doses of FF-MAS or to control solutions. The data from the control groups are presented with chromosomal status of the embryos correlated to embryo morphology. METHODS: The study includes 144 randomly selected donated human oocytes. The nucleus from each blastomere was fixed separately and fluorescence in-situ hybridization (FISH) using seven probes (13, 16, 18, 21, 22, X and Y) was performed. RESULTS: Analysis of 103 pre-embryos containing 479 blastomeres resulted in 424 blastomeres with clear FISH signals. Of these blastomeres, 55% were normal diploid and 45% were abnormal. At a pre-embryonic level, 53% were classified as normal containing >or=50% normal blastomeres while 31% of the pre-embryos were classified as uniformly normal. Abnormality rate was significantly increased in the pre-embryos with unevenly sized blastomeres and with increasing degree of fragmentation at 68 h after fertilization. Applying criteria for good embryo quality significantly increased the rate of chromosomally normal pre-embryos from 53 to 75%. CONCLUSIONS: The data demonstrate the high degree of genetic heterogeneity in a randomly selected pool of donated pre-embryos from an IVF programme. Further, we found that uniformity of blastomere size, degree of fragmentation and cleavage kinetics reflect the cytogenetic status of the pre-embryo and are therefore important in the selection of pre-embryos.

Recombinant human gonadotropins stimulate steroid and inhibin production in human granulosa cells.

OBJECTIVE: Recently pure gonadotropins have become available through recombinant technology. In parallel with ongoing clinical trials it is important to examine the effects of these new gonadotropin preparations in experimental studies in human granulosa cells. In the present study the effects of recombinant FSH (rFSH) and LH (rLH) on steroid and inhibin production were examined in human granulosa cells in culture. PATIENTS AND METHODS: Granulosa cells were obtained during the follicular phase of the menstrual cycle in seven women undergoing gynecological laparotomy and from follicles in stimulated cycles in women undergoing oocyte retrieval in connection with in vitro fertilization/embryo transfer. The granulosa cells were cultured in modified Medium 199 containing 1% fetal bovine serum for 4-8 days with and without hormones. Media were changed on alternate days and stored at -20 degrees C until analyzed for estradiol, progesterone and inhibin. RESULTS: Granulosa cells from natural cycles were highly responsive to rFSH which caused a dose-related (rFSH 0.1 to 100 ng/ml) increase in estradiol and progesterone accumulation. The maximal stimulatory effect was reached with a concentration of rFSH between 1 and 10 ng/ml. Granulosa cells from stimulated cycles responded highly to rLH in terms of increased progesterone production during the whole culture period. A maximal stimulatory effect was observed with rLH at a concentration of 0.1 ng/ml. Both types of granulosa cells responded to recombinant gonadotropins in terms of increased inhibin production. CONCLUSIONS: The present study demonstrates that granulosa cells from human ovarian follicles are highly responsive to recombinant gonadotropins as demonstrated by increased steroid and inhibin production.

Effect of growth hormone on follicular fluid androgen levels in patients treated with gonadotropins before in vitro fertilization.

Forty normally ovulating women aged 25-38 years from one private and two university in vitro fertilization (IVF) centres were used in this randomized, double-blind, parallel, placebo-controlled study to explore the effect of recombinant human growth hormone (GH) on follicular fluid (FF) levels of steroid hormones, particularly androgens. All the women had tubal factor infertility and were classified as poor responders with at least two previously performed and failed IVF treatments in which less than five oocytes had been retrieved following ovarian hyperstimulation. Growth hormone (GH 0.1 IU/kg body wt per day) or placebo was given as pretreatment during down-regulation with gonadotropin-releasing hormone agonist and during stimulation with human menopausal gonadotropin (hMG) according to the randomized protocol. Follicular fluid concentrations of steroids were measured and changes related to the levels of insulin-like growth factor I (IGF-I) and IGF binding proteins 1 and 3 and to the mode of GH administration. Pretreatment with GH, i.e. administration of GH before hMG stimulation only, caused significantly elevated follicular fluid concentrations of estrone, testosterone and dehydroepiandrosterone (DHEA) and higher values for markers of aromatase activity (ratios between estrone and androstenedione and between estradiol-17 beta and androstenedione) than in the placebo group, as well as in the two groups receiving GH during hMG stimulation. The highest values for markers of steroid sulfatase activity (ratios between DHA and DHEA sulfate and between unconjugated and conjugated estrone) were found in the patients pretreated with GH. Positive correlations were found between follicular fluid IGF-I and IGF binding protein 3 on the one hand and androgens on the other. This study showed that the administration of adjuvant GH to women who were poor responders to gonadotropins alters the endocrine/paracrine ovarian response to gonadotropins.

Effect of recombinant human gonadotrophins on oocyte meiosis and steroidogenesis in isolated pre-ovulatory rat follicles.

In order to study the effects of recombinant human luteinizing hormone (LH) and recombinant follicle stimulating hormone (FSH) on the pre-ovulatory follicle, immature pregnant mare's serum gonadotrophin-treated rats were used. Follicles were isolated by dissection and incubated for 4 h in medium 199 containing different concentrations of the gonadotrophins. In control medium the follicle-enclosed oocytes remained arrested in prophase I, but in the presence of recombinant human LH or recombinant human FSH, a concentration-dependent increase in the proportion of oocytes which underwent germinal vesicle breakdown (GVB) was seen. The lowest effective concentration of recombinant human LH was 10-50 ng/ml, and at 1000 ng/ml 97% of the oocytes underwent GVB. Recombinant human FSH was also effective, albeit slightly less so than recombinant human LH. Follicular oestradiol accumulation was stimulated up to 5-fold, both gonadotrophins showing similar effectiveness. Follicular progesterone and testosterone accumulation was markedly stimulated by recombinant human LH, giving a maximal 20-fold and 15-fold increase respectively. A 5-fold stimulation of progesterone production was found with recombinant human FSH. When submaximal concentrations of recombinant human LH and recombinant human FSH were combined, an additive effect was found on oocyte meiosis and on oestradiol and progesterone accumulation. The results are in agreement with previous studies using pituitary gonadotrophin and show that both recombinant human LH and recombinant human FSH have intrinsic effects on oocyte meiosis and steroidogenesis. It is likely that in the physiological situation in vivo, both gonadotrophins contribute to regulate these events in the peri-ovulatory period.

Steroid production in cultured thecal cells obtained from human ovarian follicles.

During follicular maturation there is a co-ordinated hormonal regulation of the theca and granulosa cells. It is generally believed that granulosa cell proliferation and differentiation are promoted mainly by follicle stimulating hormone (FSH) and that luteinizing hormone (LH) regulates the function of the theca cells. The aim of the present study was to examine the effect of LH/human chorionic gonadotrophin (HCG) on steroid production in human thecal cells. Isolated follicles (5-20 mm) were obtained during the follicular phase of the menstrual cycle in 10 women undergoing gynaecological laparotomy for reasons unrelated to ovarian pathology. The leading follicle(s) was excised and dispersed cells of the theca interna layer were isolated through combined mechanical and enzymatic techniques. The thecal cells were cultured 4-6 days with and without LH/HCG. Medium levels of androstenedione, testosterone and progesterone were measured by radioimmunoassay. Isolated thecal cells, cultured for 6 days, showed a high sensitivity to stimulation by LH/HCG. Steroid secretion was highest during days 0-2 and then declined gradually. LH/HCG stimulated steroid production in a dose-dependent way with the maximal stimulatory effect of LH at a concentration of 1-10 ng/ml, and of HCG at 0.01-0.1 IU/ml. The important question, especially in clinical situations, of the optimal level of LH for normal follicular maturation, remains to be answered. The present study is compatible with the view that thecal cell steroidogenesis in vivo is close to maximally stimulated by normal basal LH levels.

Regulation of androgen production in cultured human thecal cells by insulin-like growth factor I and insulin.

OBJECTIVES: To investigate if human thecal cells contain messenger ribonucleic acid (RNA) encoding insulin-like growth factor I (IGF-I) and insulin receptors and if IGF-I and insulin could stimulate androgen production in thecal cells. DESIGN: Poly-adenine+ RNA was extracted from fresh thecal tissue, and the expression of the genes encoding insulin and IGF-I receptors were analyzed. Isolated thecal cells were cultured 4 to 6 days with and without hormones. SETTING: Procedures were performed in a university laboratory. PATIENTS: Eight women in the follicular phase of natural cycles were undergoing gynecological laparotomy for reasons unrelated to ovarian pathology. The leading follicle(s) was excised, and dispersed cells of the theca interna layer were isolated through combined mechanical and enzymatic techniques. INTERVENTIONS: Luteinizing hormone (LH), IGF-I, and insulin were added to the cell cultures. MAIN OUTCOME MEASURE: The expression of IGF-I receptor and insulin receptor transcripts were analyzed by Northern blot. Medium levels of androstenedione and testosterone were measured by radioimmunoassay. RESULTS: In the separated thecal tissue both IGF-I receptor and insulin-receptor transcripts were detected. Insulin-like growth factor I and insulin potentiated LH-induced androgen secretion while having less pronounced effects on basal androgen production. CONCLUSION: The present study demonstrates that both insulin and IGF-I receptor genes are expressed and that insulin and IGF-I can stimulate steroid production in human thecal cells. The study provides further support for the hypothesis that IGF-I and insulin may be involved both in physiological regulation of ovarian function as well as in its pathophysiology.

Insulin-like growth factor-I stimulates amino acid accumulation in cultured human granulosa cells.

In order to study the effect of insulin-like growth factor (IGF)-I on amino acid transport, granulosa-luteal cells were obtained from stimulated cycles in women undergoing in-vitro fertilization. The cells were precultured for 1-3 days in serum-containing medium. The medium was then changed to serum-free with added IGF-I and/or human chorionic gonadotrophin (HCG). Following 24 h culture, the cellular uptake of [14C]alpha-aminoisobutyric acid AIB (0.1 mM, 0.6 mCi/ml) was studied during a 2-6 h incubation. The results showed that IGF-I (10-100 ng/ml) consistently stimulated AIB uptake to levels which were 44 +/- 7% above the control (n = 11 experiments). The stimulatory effect of IGF-I was abolished by HCG and was reduced by IGF-binding protein. In conjunction with previous findings that IGF-I stimulates the uptake and incorporation of thymidine into DNA, these results suggest that IGF-I is involved in growth of human granulosa-luteal cells.