Impact of cell loss after warming of human vitrified day 3 embryos on obstetric outcome in single frozen embryo transfers.

PURPOSE: Does cell loss (CL) after vitrification and warming (V/W) of day 3 embryos have an impact on live birth rate (LBR) and neonatal outcomes? METHOD: This retrospective analysis includes cleavage stage day 3 embryos vitrified/warmed between 2011 and 2018. Only single vitrified/warmed embryo transfers were included. Pre-implantation genetic screening, oocyte donation, and age banking were excluded from the analysis. The sample was divided into two groups: group A (intact embryo after warming) and group B (≤ 50% blastomere loss after warming). RESULTS: On the total embryos (n = 2327), 1953 were fully intact (83.9%, group A) and 374 presented cell damage (16.1%, group B). In group B, 62% (232/374) of the embryos had lost only one cell. Age at cryopreservation, cause of infertility, insemination procedure, and semen origin were comparable between the two groups. The positive hCG rate (30% and 24.3%, respectively, for intact vs CL group, p = 0.028) and LBR (13.7% and 9.4%, respectively, for intact vs CL group, p = 0.023) per warming cycle were significantly higher for intact embryos. However, LBR per positive hCG was equivalent between intact and damaged embryos (45.6% vs 38.5%, respectively, p = 0.2). Newborn measurements (length, weight, and head circumference at birth) were comparable between the two groups. Multivariate logistic regression showed that the presence of CL is not predictive for LB when adjusting for patients' age. CONCLUSIONS: LBR is significantly higher after transfer of an intact embryo compared to an embryo with CL after warming; however, neonatal outcomes are comparable between the two groups.

Expanding the time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome?

STUDY QUESTION: Is the time interval between ovulation triggering and oocyte denudation/injection associated with embryological and clinical outcome after ICSI? SUMMARY ANSWER: Expanding the time interval between ovulation triggering and oocyte denudation/injection is not associated with any clinically relevant impact on embryological or clinical outcome. WHAT IS KNOWN ALREADY: The optimal time interval between ovulation triggering and insemination/injection appears to be 38-39 h and most authors agree that an interval of >41 h has a negative influence on embryological and clinical pregnancy outcomes. However, in ART centres with a heavy workload, respecting these exact time intervals is frequently challenging. Therefore, we questioned to what extent a wider time interval between ovulation triggering and oocyte injection would affect embryological and clinical outcome in ICSI cycles. STUDY DESIGN, SIZE, DURATION: A single-centre retrospective cohort analysis was performed including 8811 ICSI cycles from 2010 until 2015. Regarding the time interval between ovulation triggering and oocyte injection, seven categories were considered: <36 h, 36 h, 37 h, 38 h, 39 h, 40 h and ≥41 h. In all cases, denudation was performed immediately prior to injection. The main outcome measures were oocyte maturation, fertilization and embryo utilization rate (embryos adequate for transfer or cryopreservation) per fertilized oocyte. Clinical pregnancy rate (CPR) and live birth rate (LBR) were considered as secondary outcomes. Utilization rate, CPR and LBR were subdivided into two groups according to the day of embryo transfer: Day 3 or Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: During the study period, oocyte retrieval was routinely performed 36 h post-triggering except in the <36 h group. The interval of <36 h occurred only if OR was carried out before the planned 36 h trigger interval and was followed by immediate injection. Only cycles with fresh autologous gametes were included. The exclusion criteria were: injection with testicular/epididymal sperm, managed natural cycles, conventional IVF, combined conventional IVF/ICSI, preimplantation genetic testing and IVM cycles. Female age, number of oocytes, pre-preparation sperm concentration, post-preparation sperm concentration and motility, day of transfer, number of embryos transferred and quality of the best embryo transferred were identified as potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Among the seven interval groups, adjusted mean maturation rates ranged from 76.4% to 83.2% and differed significantly (P < 0.001). Similarly, there was a significant difference in adjusted mean fertilization rates (range 69.2-79.3%; P < 0.001). The adjusted maturation and fertilization rates were significantly higher when denudation/injection was performed >41 h post-triggering compared to 38 h post-triggering (reference group). Oocyte denudation/injection at <36 h post-triggering had no significant effect on maturation, fertilization or embryo utilization rates compared to injection at 38 h. No effect of the time interval was observed on CPRs and LBRs, after adjusting for potential confounders. When oocyte injection was performed before 36 h the adjusted analysis showed that compared to 38 h after ovulation triggering the chance of having a live birth tends to be lower although the difference was not statistically significant (odds ratio 0.533, 95% CI: 0.252-1.126; P = 0.099). Injection ≥41 h post-triggering did not affect LBR compared to injection at 38 h post-ovulation. LIMITATIONS, REASONS FOR CAUTION: As this is a large retrospective study, the influence of uncontrolled variables cannot be excluded. These results should not be extrapolated to other ART procedures such as IVM, conventional IVF or injection with testicular/epididymal sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the optimal injection time window may be less stringent than previously thought as both embryological and clinical outcome parameters were not significantly affected in our analysis. This is reassuring for busy ART centres that might not always be able to follow strict time intervals. STUDY FUNDING/COMPETING INTEREST(S): No funding. The authors declare no conflict of interest related to the present study. TRIAL REGISTRATION NUMBER: N/A.

Is a freeze-all policy the optimal solution to circumvent the effect of late follicular elevated progesterone? A multicentric matched-control retrospective study analysing cumulative live birth rate in 942 non-elective freeze-all cycles.

STUDY QUESTION: Is late follicular elevated progesterone (LFEP) in the fresh cycle hindering cumulative live birth rates (CLBRs) when a freeze only strategy is applied? SUMMARY ANSWER: LFEP in the fresh cycle does not affect the CLBR of the frozen transfers in a freeze only approach, nor the embryo freezing rate. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which has been demonstrated to have a deleterious effect on IVF outcomes. While there is robust evidence that this elevation produces impaired endometrial receptivity, the impact on embryo quality remains a matter of debate. In particular, previous studies have shown that LFEP is associated with a hindered CLBR. However, most clinical insight on the effect of progesterone on embryo quality in terms of CLBRs have focused on embryo transfers performed after the fresh transfer, thus excluding the first embryo of the cohort. To be really informative on the possible detrimental effects of LFEP, evidence should be derived from freeze-all cycles where no fresh embryo transfer is performed in the presence of progesterone elevation, and the entire cohort of embryos is cryopreserved. STUDY DESIGN, SIZE, DURATION: This was a matched case-control, multicentre (three centres), retrospective analysis including all GnRH antagonist ICSI cycles in which a freeze all (FA) policy of embryos on day 3/5/6 of embryonic development was applied between 2012 and 2018. A total of 942 patients (471 cases with elevated P and 471 matched controls with normal P values) were included in the analysis. Each patient was included only once. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was divided according to the following P levels on the day of ovulation triggering: <1.50 ng/ml and ≥1.50 ng/ml. The matching of the controls was performed according to age (±1 year) and number of oocytes retrieved (±10%). The main outcome was CLBR defined as a live-born delivery after 24 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: The baseline characteristics of the two groups were similar. Estradiol levels on the day of trigger were significantly higher in the elevated P group. There was no significant difference in terms of fertilisation rate between the two groups. The elevated P group had significantly more cleavage stage frozen embryos compared to the normal P group while the total number of cryopreserved blastocyst stage embryos was the same. The CLBR did not differ between the two study groups (29.3% and 28.2% in the normal versus LFEP respectively, P = 0.773), also following confounder adjustment using multivariable GEE regression analysis (accounting for age at oocyte retrieval, total dose of FSH, progesterone levels on the day of ovulation trigger, day of freezing, at least one top-quality embryo transferred and number of previous IVF cycles, as the independent variables). LIMITATIONS, REASONS FOR CAUTION: This is a multicentre observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective analysis. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study demonstrating that LFEP in the fresh cycle does not hinder CLBR of the subsequent frozen cycles in a FA approach. Thus, a FA strategy circumvents the issue of elevated P in the late follicular phase. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. Throughout the study period and manuscript preparation, authors were supported by departmental funds from: Centre for Reproductive Medicine, Brussels, Belgium; Infertility Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Centro Scienze Natalità, San Raffaele Scientific Institute, Milan, Italy; and IVI-RMA, Lisbon, Portugal. E.S. has competing interests with Ferring, Merck-Serono, Theramex and Gedeon-Richter outside the submitted work. E.P. reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from MSD and grants from IBSA outside the submitted work. All the other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Chromosomal abnormalities after ICSI in relation to semen parameters: results in 1114 fetuses and 1391 neonates from a single center.

STUDY QUESTION: Is there a relationship between karyotype abnormalities in fetuses and children conceived by ICSI and their father's semen parameters? SUMMARY ANSWER: The de novo chromosomal abnormality rate in pre- and postnatal karyotypes of ICSI offspring was higher than in the general population and related to fathers' sperm parameters. WHAT IS KNOWN ALREADY: Several studies have reported a higher rate of de novo chromosomal anomalies in ICSI fetuses but recent data from large cohorts are limited. Overall, reported prevalences of non-inherited karyotype aberrations are increased in fetuses conceived after ICSI and vary between 1.6% and 4.2%. Only a few studies focus on the relation between karyotype anomalies in ICSI offspring and semen parameters of their fathers. Furthermore, an increased incidence of abnormal karyotypes in ICSI neonates has been described, but the rates vary widely across studies. STUDY DESIGN, SIZE, DURATION: We report on karyotype results from prenatal testing by means of chorionic villus sampling and amniocentesis and results from postnatal blood sampling in offspring conceived by ICSI in a single center. Ongoing pregnancies resulting from an oocyte retrieval between January 2004 and December 2012 and after transfer of fresh ICSI embryos obtained using ejaculated or non-ejaculated sperm (fresh or frozen-thawed) were considered. Pregnancies following frozen embryo transfer, oocyte or sperm donation, IVF, preimplantation genetic testing and IVM were excluded. All abnormal prenatal results after sampling are reported irrespective of the outcome of the pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS: From the 4816 ongoing ICSI pregnancies, information on pregnancy outcome was available for 4267 pregnancies. Prenatal testing was performed in 22.3% of the pregnancies, resulting in a diagnosis in 1114 fetuses. A postnatal karyotype was obtained in 29.4% of the pregnancies in which no invasive prenatal diagnosis was performed, resulting in a total of 1391 neonates sampled. The prevalence of chromosomal anomalies according to maternal age and semen quality was analyzed with logistic regression. For definitions of normal semen quality, the World Health Organization reference values for human semen characteristics were adopted. MAIN RESULTS AND THE ROLE OF CHANCE: An abnormal fetal karyotype was found in 29 singletons and 12 multiples (41/1114; 3.7%; 95% CI 2.7-4.9%): 36 anomalies were de novo (3.2%; 95% CI 2.3-4.4), either numerical (n = 25), sex (n = 6) or structural (n = 5), and five were inherited. Logistic regression analysis did not show a significant association between maternal age and a de novo chromosomal fetal abnormality (odds ratio (OR) 1.05; 95% CI 0.96-1.15; P = 0.24). In all but one case, fetuses with an abnormal karyotype were conceived by ICSI using ejaculated sperm.Abnormal karyotypes were found in 14 (1.0%; 95% CI 0.6-1.7) out of 1391 postnatal samples of children born after ICSI who were not tested prenatally: 12 were de novo anomalies and two were inherited balanced karyotypes. The 14 abnormal karyotypes were all found in children born after ICSI using ejaculated sperm.The odds of a de novo karyotype aberration increased with maternal age when combining pre- and postnatal data (OR 1.11; 95% CI 1.04-1.19). A higher rate of de novo chromosomal abnormalities was found in fetuses and children of couples with men having a sperm concentration <15 million/ml (adjusted OR (AOR) 2.10; 95% CI 1.14-3.78), sperm concentration <5 million/ml (AOR 1.9; 95% CI 1.05-3.45) and total sperm count <10 million (AOR 1.97; 95% CI 1.04-3.74). LIMITATIONS, REASONS FOR CAUTION: We cannot exclude that the observation of a higher prevalence of karyotype anomalies in ICSI offspring compared to literature data in the general population is due to enhanced surveillance after ART given the lack of a control group. Although we did not find more chromosomal anomalies after ICSI with non-ejaculated sperm, the small numbers do not allow firm conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The observed increased risk of a de novo karyotype anomaly after ICSI conception in couples with poor sperm warrants continued counseling toward prenatal testing.The current and widespread use of innovative non-invasive prenatal testing will result in larger datasets, adding to a balanced estimation of the prevalence of karyotype anomalies in ICSI offspring. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Methusalem grants issued by the Vrije Universiteit Brussel. All authors declared no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: N/A.

Do we need to measure progesterone in oocyte donation cycles? A retrospective analysis evaluating cumulative live birth rates and embryo quality.

STUDY QUESTION: Does late follicular-phase elevated serum progesterone (LFEP) during ovarian stimulation for oocyte donation have an impact on embryo quality (EQ) and cumulative live birth rate (CLBR)? SUMMARY ANSWER: LFEP does not have an influence on EQ nor CLBR in oocyte donation cycles. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which, when elevated during the follicular phase, has been demonstrated to have a deleterious effect in autologous fresh IVF outcomes. While there is robust evidence that this elevation results in impaired endometrial receptivity, the impact on EQ remains a matter of debate. The oocyte donation model is an excellent tool to assess the effects of LFEP on EQ from those on endometrium receptivity separately. Previous studies in oocyte donation cycles investigating the influence of elevated P on pregnancy outcomes in oocyte recipients showed conflicting results. STUDY DESIGN, SIZE, DURATION: This is a retrospective analysis including all GnRH antagonist down-regulated cycles for fresh oocyte donation taking place in a tertiary referral university hospital between 2010 and 2017. A total of 397 fresh donor-recipient cycles were included. Each donor was included only once in the analysis and could be associated to a single recipient. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was stratified according to serum P levels of ≤1.5 and >1.5 ng/mL on the day of ovulation triggering. The primary endpoint of the study was the top-quality embryo rate on Day 3, and the secondary outcome measure was CLBR defined as a live-born delivery beyond 24 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Three hundred ninety-seven fresh oocyte donation cycles were included in the analysis, of which 314 (79%) had a serum P ≤ 1.5 ng/mL and 83 (20.9%) had a serum P > 1.5 ng/mL. The average age of the oocyte donors was 31.4 ± 4.7 and 29.9 ± 4.5 years, respectively, for normal and elevated P (P = 0.017). The mean number of oocytes retrieved was significantly higher in the elevated P group with 16.6 ± 10.6 vs 11.5 ± 6.9 in the P ≤ 1.5 group (P < 0.001).In parallel, the total number of embryos on Day 3, as well as the number of good-quality embryos at this stage, was significantly higher in the elevated P group (6.6 ± 5.6 vs 4.15 ± 3.5 and 8.7 ± 6.3 vs 6.1 ± 4.4; respectively, P < 0.001). However, maturation and fertilization rates did not vary significantly between the two study groups and neither did the top- and good-quality embryo rate and the embryo utilization rate, all evaluated on Day 3 (P = 0.384, P = 0.405 and P = 0.645, respectively). A multivariable regression analysis accounting for P groups, age of the donor, number of retrieved oocytes and top-quality embryo rate as potential confounders showed that LFEP negatively influenced neither the top-quality embryo rate nor the CLBR. LIMITATIONS, REASONS FOR CAUTION: This is an observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective trial. Furthermore, this study was focused on oocyte donation cycles and hence the results cannot be generalized to the entire infertile population. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study providing evidence that LFEP does not influence CLBR and is adding strong evidence to the existing literature that LFEP does not harm EQ in oocyte donation programs. STUDY FUNDING/COMPETING INTERESTS: Not applicable.

Clinical performance of a specific granulocyte colony stimulating factor ELISA to determine its concentration in follicular fluid as a predictor of implantation success during in vitro fertilization.

This study aimed to demonstrate the clinical performance of an ultra-sensitive follicular fluid (FF) granulocyte colony stimulating factor (G-CSF) immunoassay to confirm previous work, indicating a correlation between FF G-CSF concentration and live birth potential of the corresponding embryo after in vitro fertilization. This study was a noninterventional, prospective, diagnostic clinical multicentric study conducted between August 2012 and January 2014 with 396 single embryo transfers (SETs) from 278 subjects. During oocyte retrieval, FF was individually collected. Embryo morphology and implantation success were evaluated. The implantation success rate in the high G-CSF group (32.3%) was higher than the overall rate (27.5%). Similarly, for embryos with optimal morphology, implantation success rates were highest among those in the high G-CSF concentration category (34.5%) compared with low (19.6%) and intermediate (29.8%) G-CSF concentration categories. Significant differences in mean G-CSF concentrations were observed between the study sites. To minimize bias, analyses were repeated using data from the center with the largest number of SETs. In alignment with the overall analysis, this center demonstrated a 43% greater probability of implantation for optimal embryos with high G-CSF compared to the general implantation rate among optimal embryos and a 327% increase compared with the implantation rate of optimal embryos with low G-CSF.

Influence of ultra-low oxygen (2%) tension on in-vitro human embryo development.

STUDY QUESTION: Is a reduction in the oxygen tension from 5 to 2% during extended culture from Day 3 onwards beneficial for human blastocyst development in vitro? SUMMARY ANSWER: A reduction in oxygen concentration from 5 to 2% O2 after Day 3 did not improve embryo development, quality and utilization rate. WHAT IS KNOWN ALREADY: The human embryo leaves the fallopian tube to reach the uterine cavity around Day 3-4 post-ovulation. As the oxygen concentration ranges from 5 to 7% in the fallopian tube and decreases to 2% in the uterus, reducing the oxygen tension during extended culture from Day 3 onwards seems more physiological. We aim to mimic the in-vivo environment during in-vitro embryo culture. Therefore, we compared the effect of extended culture performed at 5% (control arm) or 2% oxygen (O2; study arm) tension on blastocyst formation and quality. STUDY DESIGN, SIZE, DURATION: Between December 2016 and September 2017, in two prospective studies, sibling embryos were randomized on Day 3 to either 5% O2 (control) or 2% O2 (study) for extended culture. In the control arms of both studies 1 and 2, the dishes with blastocyst medium were pre-equilibrated overnight in 5% O2, 6% CO2 and 89% N2 at 37°C. In the 2% study groups, the overnight pre-equilibration of blastocyst media was performed in either 2% O2 (study 1, 99 cycles) or 5% O2 (study 2, 126 cycles). The latter provides a gradual transition from 5 to 2% O2 environment for the study arm. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Embryo culture until Day 3 was always performed in 5% O2; if at least four embryos of moderate to excellent quality were obtained on Day 3, the sibling embryos were randomized to either 5% O2 or 2% O2 for extended culture. The endpoints were embryo development and quality on Day 5/6 and the utilization rate (embryos transferred and cryopreserved). Statistical analysis was performed using the chi-square test, a P-value of <0.05 was considered significantly different. MAIN RESULTS AND THE ROLE OF CHANCE: In study 1, 811 embryos were randomized on Day 3: 405 to the 2% O2 and 406 to the 5% O2 condition. No differences were observed in the blastulation rate (68.6 versus 71.9%; P = 0.319) and the proportion of good quality blastocysts on Day 5 (55.8 versus 55.2%; P = 0.888), nor in the utilization rate (53.1 versus 53.2%; P = 1.000). In study 2, 1144 embryos were randomized: 572 in each arm. Similarly, no significant difference was demonstrated in terms of the blastulation rate (63.6 versus 64.7%; P = 0.758), the proportion of good quality blastocysts (46.9 versus 48.8%; P = 0.554) or the utilization rate (49.8 versus 48.1%; P = 0.953). LIMITATIONS, REASON FOR CAUTION: This study evaluated embryo development only until Day 5/6. The effect of oxidative stress on the developing embryo may only become evident at later stages (i.e. during implantation) and should therefore be studied in an RCT. The question also remains as to whether the switch to ultra-low oxygen tension from Day 4 onwards, when the embryo arrives in the uterus in vivo, would be preferential. WIDER IMPLICATIONS OF THE FINDINGS: Based on the present study results, there is no benefit in lowering the oxygen tension from 5 to 2% from Day 3 onwards during extended human embryo culture. STUDY FUNDING/COMPETING INTEREST(s): No funding was received for this study and the authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Predicting suboptimal oocyte yield following GnRH agonist trigger by measuring serum LH at the start of ovarian stimulation.

STUDY QUESTION: Are the LH levels at the start of ovarian stimulation predictive of suboptimal oocyte yield from GnRH agonist triggering in GnRH antagonist down-regulated cycles? SUMMARY ANSWER: LH levels at the start of ovarian stimulation are an independent predictor of suboptimal oocyte yield following a GnRH agonist trigger. WHAT IS KNOWN ALREADY: A GnRH agonist ovulation trigger may result in an inadequate oocyte yield in a small subset of patients. This failure can range from empty follicle syndrome to the retrieval of much fewer oocytes than expected. Suboptimal response to a GnRH agonist trigger has been defined as the presence of circulating LH levels <15 IU/l 12 h after triggering. It has been shown that patients with immeasurable LH levels on trigger day have an up to 25% risk of suboptimal response. STUDY DESIGN, SIZE, DURATION: In this retrospective cohort study, all patients (n = 3334) who received GnRH agonist triggering (using Triptoreline 0.2 mg) for final oocyte maturation undergoing a GnRH antagonist cycle in our centre from 2011 to 2017 were included. The primary outcome of the study was oocyte yield, defined as the ratio between the total number of collected oocytes and the number of follicles with a mean diameter >10 mm prior to GnRH agonist trigger. PARTICIPANTS/MATERIALS, SETTING, METHODS: The endocrine profile of all patients was studied at initiation as well as at the end of ovarian stimulation. In order to evaluate whether LH levels, not only at the end but also at the start, of ovarian stimulation predicted oocyte yield, we performed multivariable regression analysis adjusting for the following confounding factors: female age, body mass index, oral contraceptives before treatment, basal and trigger day estradiol levels, starting FSH levels, use of highly purified human menopausal gonadotrophin and total gonadotropin dose. Suboptimal response to GnRH agonist trigger was defined as <10th percentile of oocyte yield. MAIN RESULTS AND THE ROLE OF CHANCE: The average age was 31.9 years, and the mean oocyte yield was 89%. The suboptimal response to GnRH agonist trigger cut-off (<10th percentile) was 45%, which was exhibited by 340 patients. Following confounder adjustment, multivariable regression analysis showed that LH levels at the initiation of ovarian stimulation remained an independent predictor of suboptimal response even in the multivariable model (adjusted OR 0.920, 95% CI 0.871-0.971). Patients with immeasurable LH levels at the start of stimulation (<0.1 IU/l) had a 45.2% risk of suboptimal response, while the risk decreased with increasing basal LH levels; baseline circulating LH <0.5 IU/L, <2 IU/L and <5 IU/L were associated with a 39.1%, 25.2% and 13.6% risk, respectively. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is its retrospective design. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest study of GnRH agonist trigger cycles only, since most of the previous research on the predictive value of basal LH levels was performed in dual trigger cycles. LH values should be measured prior to start of ovarian stimulation. In cases where they are immeasurable, suboptimal response to GnRH agonist trigger can be anticipated, and an individualized approach is warranted. STUDY FUNDING/COMPETING INTEREST(S): There was no funding and no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Factors influencing the clinical outcome of preimplantation genetic testing for polycystic kidney disease.

STUDY QUESTION: What are the factors influencing the success rate for couples undergoing preimplantation genetic testing (PGT) for polycystic kidney disease (PKD)? SUMMARY ANSWER: In our study cohort, the live birth delivery rate is significantly associated with female age while the male infertility accompanying autosomal dominant PKD (ADPKD) does not substantially affect the clinical outcome. WHAT IS KNOWN ALREADY: While women with ADPKD have no specific fertility problems, male ADPKD patients may present with reproductive system abnormalities and infertility. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study involves 91 PGT cycles for PKD for 43 couples (33 couples for PKD1, 2 couples for PKD2 and 8 couples for autosomal recessive PKD (ARPKD)) from January 2005 until December 2016 with follow-up of transfers until end of 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixteen single-cell clinical tests for PKD based on multiplex PCR of short tandem repeat markers, with or without a specific mutation were developed and applied for diagnosis of 584 Day 3 cleavage stage embryos. In 18 couples, the male partner was affected with ADPKD (=Group A) and 12 of them had a documented infertility status. Group A underwent 52 cycles to oocyte retrieval. For 18 other couples, the female partner was affected with ADPKD (=Group B) and four male partners from this group had a documented history of infertility. This group underwent 31 cycles to OR. MAIN RESULTS AND THE ROLE OF CHANCE: Genetic analysis resulted in 545 embryos (93.3%) with a diagnosis, of which 215 (36.8%) were genetically transferable. Transfer of 74 embryos in 53 fresh cycles and of 34 cryopreserved embryos in 33 frozen-warmed embryo transfer cycles resulted in a live birth delivery rate of 38.4% per transfer with 31 singleton live births, two twin live births and one ongoing pregnancy. The observed cumulative delivery rate was 57.8% per couple after five treatment cycles. Thirty cryopreserved embryos still remain available for transfer. The clinical pregnancy rate per transfer (fresh + frozen; 45.9% in group A versus 60.0% in group B, P < 0.05) and the live birth delivery rate per transfer (fresh + frozen; 27.0% in group A versus 42.9% in group B, P < 0.05) was significantly lower for couples with the male partner affected with ADPKD compared with couples with the female partner affected with ADPKD. However, a multivariate logistic regression analysis showed that only female age was associated with live birth delivery rate (odds ratio = 0.87; 95% CI: 0.77-0.99; P = 0.032). LIMITATIONS, REASONS FOR CAUTION: This study is based on retrospective data from a single centre with Day 3 one-cell and two-cell biopsy. Further analysis of a larger cohort of PKD patients undergoing PGT is required to determine the impact of male infertility associated with ADPKD on the cumulative results. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge about factors affecting the clinical outcome after PGT can be a valuable tool for physicians to counsel PKD patients about their reproductive options. Males affected with ADPKD who suffer from infertility should be advised to seek treatment in time to improve their chances of conceiving a child. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

BMP4 plays a role in apoptosis during human preimplantation development.

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra-embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4-treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4-treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.

Cumulus-corona gene expression analysis combined with morphological embryo scoring in single embryo transfer cycles increases live birth after fresh transfer and decreases time to pregnancy.

PURPOSE: Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies. METHODS: A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and ß2M). Patients (n = 62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes. RESULTS: Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p < 0.001) and day 5 (43% and 39%: p = 0.022 and p = 0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3. CONCLUSION: Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy. TRIAL REGISTRATION NUMBER: This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).

Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality?

STUDY QUESTION: Is elevated late-follicular phase progesterone (EP) associated with a deleterious impact on embryo quality (EQ) and cumulative live birth rates (LBRs)? SUMMARY ANSWER: EP was associated with a decrease in embryo utilization and cumulative LBRs. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which adversely affects IVF pregnancy outcomes. However, evidence regarding a potential association between EP an EQ is lacking. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of all GnRH antagonist down-regulated ICSI cycles followed by a fresh embryo transfer (ET) between 2010 and 2015 was performed. The sample was stratified according to the following P levels on the day of ovulation triggering: ≤0.50, 0.51-1.49 and ≥1.50 ng/ml. The primary outcomes were embryo utilization rates (number of embryos transferred or cryopreserved) and cumulative LBR, defined as the occurrence of the first live-birth after either the fresh or one of the subsequent frozen ET. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, 3400 cycles were included in the analysis, using multivariable regression to account for potential confounding. MAIN RESULTS AND THE ROLE OF CHANCE: Female age and the number of oocytes retrieved increased significantly with increasing serum P values. Utilization rates decreased linearly as P increased for Day 3 embryos (72.3, 63.0 and 45.4%, respectively), while for Day 5 embryos only the EP group was associated with a significant decrease (48.8, 47.8 and 38.8%, respectively). EP was also associated with decreased fresh and cumulative LBRs. LIMITATIONS REASONS FOR CAUTION: The main limitations of this study were its retrospective nature and the fact that it was restricted to GnRH antagonist cycles. WIDER IMPLICATIONS OF THE FINDINGS: These results raise the question whether EP may also be associated with a decrease in cumulative pregnancy outcomes by increasing embryo wastage. Further studies may evaluate the potential benefit of additional measures besides the freeze-all strategy to avoid this issue, such as lowering the stimulation dose or applying a step-down protocol. STUDY FUNDING/COMPETING INTEREST(S): None.

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

STUDY QUESTION: Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? SUMMARY ANSWER: A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. WHAT IS KNOWN ALREADY: IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. STUDY DESIGN, SIZE, DURATION: A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. MAIN RESULTS AND THE ROLE OF CHANCE: PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. LIMITATIONS REASONS FOR CAUTION: The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. STUDY FUNDING/COMPETING INTEREST(S): IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Closed oocyte vitrification and storage in an oocyte donation programme: obstetric and neonatal outcome.

STUDY QUESTION: Does closed oocyte vitrification in an oocyte donation programme have an impact on obstetric and neonatal outcome? SUMMARY ANSWER: Obstetric and neonatal outcomes after closed system vitrification of donor oocytes appear to be reassuring. WHAT IS KNOWN ALREADY: The use of fresh oocytes has not been proved to be superior to the use of vitrified donor oocytes in terms of survival, embryo development and clinical pregnancies. Those studies used open devices to prove the non-superiority. Very limited information is available on the comparison of open and closed devices, and the results for survival, embryo development and pregnancy outcomes are conflicting. Data on obstetric and neonatal outcome from vitrified oocytes are scarce. Only one large report is available after the use of donor oocytes vitrified with an open device. STUDY DESIGN, SIZE, DURATION: Retrospective observational study performed at the Centre for Reproductive Medicine, UZ Brussel, Belgium. All 117 oocyte recipient cycles between March 2010 and August 2014 with the use of a closed vitrification device and leading to a pregnancy beyond 20 weeks were included in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All recipient warming cycles with a pregnancy beyond 20 weeks from vitrified donor oocytes: results from the fresh embryo transfers. MAIN RESULTS AND THE ROLE OF CHANCE: For 117 recipient cycles, a total of 793 oocytes were warmed of which 657 (82.8%) survived and 499 (76.0%) were fertilized. Nineteen single and 98 double embryo transfers led to 95 singleton and 22 twin pregnancies. Hypertensive disorders, haemorrhages and gestational diabetes were reported in 22/112 (19.6%), 30/112 (26.8%) and 13/112 (11.6%) of the pregnancies, respectively. No major adverse neonatal outcomes were observed. Congenital malformations were observed in 11 out of 139 children; for one an elective termination was performed at 25 weeks. LIMITATIONS, REASONS FOR CAUTION: Since March 2010, almost all oocytes for donation are vitrified in our centre. Therefore, no recent data are available to control the outcomes of fresh oocyte donations. WIDER IMPLICATIONS OF THE FINDINGS: The reassuring results obtained in the current study show that closed system vitrification devices for donor oocytes may be used as an alternative to open devices which have been linked to possible cross-contamination issues. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

Neonatal health including congenital malformation risk of 1072 children born after vitrified embryo transfer.

STUDY QUESTION: Does vitrification of Day 3 and Day 5 embryos adversely affect birth outcomes of singletons and twins in comparison with peers born after fresh embryo transfer? SUMMARY ANSWER: Neonatal health parameters, including the prevalence of congenital malformations, in singletons and twins born after embryo vitrification are similar to or slightly better than after fresh embryo transfer. WHAT IS ALREADY KNOWN: Although vitrification, rather than slow-freezing, of embryos is routine practice nowadays, convincing evidence regarding the safety for the offspring is sparse. Literature data comprise results from mostly small-sized studies or studies including only Day 3 or only Day 5 vitrified embryo transfers. Overall, better or comparable perinatal outcomes, in terms of higher birthweight and lower risk for small-for-gestational age or for low birthweight, have been reported for singletons born after vitrified embryo transfer compared with fresh embryo transfer. According to the single available study with sufficient sample size, the congenital malformation rate was found to be comparable after vitrified and fresh embryo transfers. STUDY DESIGN, SIZE, DURATION: Data were collected from 960 cycles after transfer of embryos vitrified on Day 3 (n = 457) or Day 5 (n = 503) and from 1644 cycles after fresh embryo transfer on Day 3 (n = 853) or Day 5 (n = 791), performed between 2008 and 2013 at the Centre for Reproductive Medicine of the university hospital UZ Brussel. Outcome measures were neonatal health in terms of birthweight, small-for-gestational age, prematurity rate, perinatal death and major/minor/total malformation rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Perinatal health parameters of 11 stillborns and 1061 live borns (827 singletons and 234 twins) in the vitrified group and of 28 stillborns and 1838 live borns (1374 singletons and 464 twins) in the fresh embryo group are reported. Within 3 months after birth, children in the two study groups were assessed clinically with special attention to congenital malformations by a paediatrician blinded to the type of embryo transfer. Data were analysed by multiple linear and logistic regression, adjusted for treatment variables and maternal characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Mothers to infants in the vitrified group were on average slightly older and more often suffering from pregnancy-related hypertensive disorders than mothers to infants in the fresh transfer group. Singletons born after vitrification showed a higher birthweight standard deviation score (SDS) (-0.4 versus -0.7; 95% confidence interval (CI): 0.0-0.3, P = 0.001) and a lower small-for-gestational age rate (AOR: 0.55; 95% CI: 0.34-0.90) in comparison with peers born after fresh embryo transfer. Preterm birth rate and perinatal death rate were comparable between the two groups (AOR: 0.91; 95% CI: 0.57-1.43 and AOR: 0.97; 95% CI: 0.40-2.36). In twins, neonatal outcomes including birthweight SDS, small-for-gestational age and prematurity rates were comparable in the vitrified and the fresh groups, when adjusted for confounders. Furthermore, the rate of major congenital malformations in live borns was comparable between the vitrified group and the fresh group, both in singletons (2.6 versus 2.8%; AOR: 0.91; 95% CI: 0.47-1.78) and in twins (2.4 versus 2.7%; AOR: 0.51; 95% CI: 0.05-5.72). Also, the total malformation rate in the vitrified group (3.4%; 95% CI: 2.4-4.8) did not differ from the rate in the fresh embryo group (3.9%; 95% CI: 3.1-5.0). The embryonic stage at vitrification or fresh transfer (cleavage-stage embryo or blastocyst) did not influence the birth characteristics or malformation rate. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study is the rather small twin group. Therefore, the outcome results for twins should be interpreted cautiously. WIDER IMPLICATIONS OF THE FINDINGS: This study provides evidence that transfer of vitrified Day 3 and Day 5 embryos does not adversely affect the neonatal health of the offspring in comparison with transfer of fresh embryos. Furthermore, neonatal outcomes were not different after transfer of vitrified blastocysts compared with transfer of vitrified cleavage-stage embryos. STUDY FUNDING/COMPETING INTERESTS: Educational grants for establishing and organizing the data collection have come from IBSA, Ferring, Organon, Shering-Plough and Merck. Merck Belgium funded the data collection for outcomes after vitrification between 2012 and 2015. All co-authors, except M.B., declared no conflict of interest. M.B. has received consultancy fees from Organon, Serono Symposia and Merck.

Open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study.

STUDY QUESTION: Is the survival of donor oocytes with the CryotopSC device superior to the survival with the closed CBSvit device? SUMMARY ANSWER: The CryotopSC device and the CBSvit device showed similar survival rates. WHAT IS KNOWN ALREADY: Health authorities are cautious about possible cross contamination during liquid nitrogen storage or handling when working with open vitrification devices. At present, the use of open devices is still allowed since little information is available on the efficiency of closed devices. STUDY DESIGN, SIZE, DURATION: A prospective randomized sibling oocyte study was performed in the Centre for Reproductive Medicine (UZBrussel) between January 2014 and July 2015. The survival after warming and the embryological outcome of donor oocytes vitrified using two devices was compared: the CBSvit device (closed vitrification and closed storage) and the CryotopSC device (open vitrification and closed storage). A difference of 10% was defined to prove the superiority of the CryotopSC device. In total, 250 warmed oocytes were needed in each arm. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes from 48 donors were included in the study: 253 vitrified with the CBSvit device and 257 with the CryotopSC device. Equal numbers of oocytes from both devices and originated from the same donor cycle were allocated to each of 78 recipients, in order to exclude donor and recipient (male factor) effects. MAIN RESULTS AND THE ROLE OF CHANCE: There were no differences found between the CBSvit and the CryotopSC in terms of survival after warming (93.7 versus 89.9%) or fertilization per injected oocyte (74.3 versus 81.4%). The degeneration rate after ICSI was significantly higher for the CBSvit device: 11.4 versus 6.1% (P = 0.041). A significantly higher number of zygotes in the CryotopSC group finished their first mitosis 25-27 h post-injection (34.1 versus 52.1%, P = 0.001). On Day 3, the overall embryo quality distribution did not vary between groups, but a significantly higher cell number was obtained in the CryotopSC device: 6.8 ± 2.8 versus 7.6 ± 2.8 (P = 0.01). The utilization rate per mature oocyte, per surviving oocyte or per fertilized oocyte did not differ. The embryos with the highest quality were selected for transfer on Day 3. The clinical pregnancy rate per transfer cycle was 36.5%. LIMITATIONS, REASONS FOR CAUTION: The results of this study should not be extrapolated to other female groups, since oocytes from young fertile donors were used in this study. WIDER IMPLICATIONS OF THE FINDINGS: In many countries, the use of open devices is still allowed due to the limited reports on the efficiency of closed devices. Knowing the caution of health authorities about the use of open devices, there is an urgent need for efficiency studies with closed devices. The results obtained in the current study shows the efficiency of a safe closed vitrification device, leaving behind any concern about possible cross contamination during handling or storage. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: NCT01952184. TRIAL REGISTRATION DATE: 24 September 2013. DATE OF FIRST PATIENT'S ENROLMENT: 23 January 2014.

Do ARTs affect the incidence of monozygotic twinning?

STUDY QUESTION: Does the manipulation of gametes or embryos during ARTs increase the risk for monozygotic twinning (MZT)? SUMMARY ANSWER: Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. WHAT IS KNOWN ALREADY: Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. STUDY DESIGN, SIZE, DURATION: All clinical pregnancies after single ET carried out in our centre between 2004 and 2013 (n = 6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. MAIN RESULTS AND THE ROLE OF CHANCE: The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29-0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36-5.34; aOR 2.05, 95% CI 1.29-3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. LIMITATION, REASONS FOR CAUTION: This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. WIDER IMPLICATION OF THE FINDINGS: Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. STUDY FUNDING/COMPETING INTERESTS: No external funding was used for this study. There are no conflicts of interest.

Semen quality of young adult ICSI offspring: the first results.

STUDY QUESTION: What is the semen quality of young adult men who were conceived 18-22 years ago by ICSI for male infertility? SUMMARY ANSWER: In this cohort of 54 young adult ICSI men, median sperm concentration, total sperm count and total motile sperm count were significantly lower than in spontaneously conceived peers. WHAT IS KNOWN ALREADY: The oldest ICSI offspring cohort worldwide has recently reached adulthood. Hence, their reproductive health can now be investigated. Since these children were conceived by ICSI because of severe male-factor infertility, there is reasonable concern that male offspring have inherited the deficient spermatogenesis from their fathers. Previously normal pubertal development and adequate Sertoli and Leydig cell function have been described in pubertal ICSI boys; however, no information on their sperm quality is currently available. STUDY DESIGN, SIZE, DURATION: This study was conducted at UZ Brussel between March 2013 and April 2016 and is part of a large follow-up project focussing on reproductive and metabolic health of young adults, between 18 and 22 years and conceived after ICSI with ejaculated sperm. Results of both a physical examination and semen analysis were compared between young ICSI men being part of a longitudinally followed cohort and spontaneously conceived controls who were recruited cross-sectionally. PARTICIPANTS/MATERIALS, SETTING, METHOD: Results of a single semen sample in 54 young adult ICSI men and 57 spontaneously conceived men are reported. All young adults were individually assessed, and the results of their physical examination were completed by questionnaires. Data were analysed by multiple linear and logistic regression, adjusted for covariates. In addition, semen parameters of the ICSI fathers dating back from their ICSI treatment application were analysed for correlations. MAIN RESULTS AND THE ROLE OF CHANCE: Young ICSI adults had a lower median sperm concentration (17.7 million/ml), lower median total sperm count (31.9 million) and lower median total motile sperm count (12.7 million) in comparison to spontaneously conceived peers (37.0 million/ml; 86.8 million; 38.6 million, respectively). The median percentage progressive and total motility, median percentage normal morphology and median semen volume were not significantly different between these groups. After adjustment for confounders (age, BMI, genital malformations, time from ejaculation to analysis, abstinence period), the statistically significant differences between ICSI men and spontaneously conceived peers remained: an almost doubled sperm concentration in spontaneously conceived peers in comparison to ICSI men (ratio 1.9, 95% CI 1.1-3.2) and a two-fold lower total sperm count (ratio 2.3, 95% CI 1.3-4.1) and total motile count (ratio 2.1, 95% CI 1.2-3.6) in ICSI men compared to controls were found. Furthermore, compared to men born after spontaneous conception, ICSI men were nearly three times more likely to have sperm concentrations below the WHO reference value of 15 million/ml (adjusted odds ratio (AOR) 2.7; 95% CI 1.1-6.7) and four times more likely to have total sperm counts below 39 million (AOR 4.3; 95% CI 1.7-11.3). In this small group of 54 father-son pairs, a weak negative correlation between total sperm count in fathers and their sons was found. LIMITATIONS, REASONS FOR CAUTION: The main limitation is the small study population. Also, the results of this study where ICSI was performed with ejaculated sperm and for male-factor infertility cannot be generalized to all ICSI offspring because the indications for ICSI have nowadays been extended and ICSI is also being performed with non-ejaculated sperm and reported differences may thus either decrease or increase. WIDER IMPLICATIONS OF THE FINDINGS: These first results in a small group of ICSI men indicate a lower semen quantity and quality in young adults born after ICSI for male infertility in their fathers. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts, all issued by the Vrije Universiteit Brussel (VUB). All co-authors except M.B. and H.T. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough and Merck for establishing the database for follow-up research and organizing the data collection. The institution of H.T. has received research grants from the Research Fund of Flanders (FWO), an unconditional grant from Ferring for research on testicular stem cells and research grants from Ferring, Merck, MSD, Roche, Besins, Goodlife and Cook for several research projects in female infertility. H.T. has received consultancy fees from Finox, Abbott and ObsEva for research projects in female infertility.

Frozen-thawed embryo transfers in natural cycles with spontaneous or induced ovulation: the search for the best protocol continues.

STUDY QUESTION: Is natural cycle frozen-thawed embryo transfer (NC-FET) associated with better clinical pregnancy rates (CPR) when compared to modified natural cycle frozen-thawed embryo transfer (mNC-FET)? SUMMARY ANSWER: NC-FET is associated with a higher CPR compared to mNC-FET. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the impact of hCG triggering on clinical outcomes after frozen-thawed embryo transfer (FET), and information on the effect of luteal phase support (LPS) is lacking. STUDY DESIGN, SIZE, DURATION: This retrospective study included all (n = 2353) consecutive cycles with FET of vitrified cleavage and blastocyst stage embryos warmed between January 2010 and April 2015 in a tertiary centre. The FET cycles were grouped by type as follows: NC (n = 501), NC + LPS (n = 828) or mNC + LPS (n = 1024). Artificial cycles were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed mixed-effect multilevel multivariable regression analysis to account for the clustering of FETs using embryos derived from the same patient and/or ovarian stimulation cycle. Adjustment for the following potential confounders was also performed: female age at oocyte retrieval, number of oocytes retrieved, fresh cycle pregnancy outcome, embryo transfer rank, number of embryos transferred, embryo stage and grade and endometrial thickness. Bonferroni adjustment for multiple comparisons was performed whenever indicated. MAIN RESULTS AND THE ROLE OF CHANCE: The unadjusted CPR per cycle was significantly higher in the NC-FET group (46.9%) when compared with the mNC-FET + LPS groups (29.7%, P < 0.001) but not the NC-FET + LPS group (39.9%, P = 0.069). The lower clinical performance of mNC-FET + LPS remained significant even after adjusting for potential confounders [adjusted odds ratio (95% CI) compared to the NC-FET groups: 2.18 (1.64-2.90) and 1.67 (1.31-2.12) for the NC-FET and NC-FET + LPS groups, respectively]. A sensitivity analysis restricting the sample only to the first FET performed by the couple in our centre was also performed. The predicted CPR in this multivariable logistic regression model remained significantly higher in the NC-FET (53.9%) and NC-FET + LPS (44.9%) groups when compared to mNC-FET + LPS (34.2%, all Bonferroni-adjusted pairwise comparisons with P ≤ 0.01). LIMITATIONS, REASONS FOR CAUTION: The interpretation of the findings of this study is limited by the retrospective nature of the analysis and the potential for unmeasured confounding. WIDER IMPLICATIONS OF THE FINDINGS: The use of mNC-FET, using hCG triggering or progesterone supplementation, should be reconsidered in light of the potential negative effect on pregnancy outcome. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.