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Research question Is implantation impaired in patients with endometriosis undergoing IVF and intracytoplasmatic sperm injection (ICSI) cycles? Design A retrospective matched cohort study was carried out on IVF/ICSI cycles with fresh single embryo transfer at the Department of Assisted Reproductive Medicine, Ghent University Hospital, Belgium, between July 2015 and August 2017 (n = 1053). A total of 118 endometriosis cases were matched 1:1 to 118 couples diagnosed with male subfertility and stratified by embryo quality (identical ALPHA grading categories), female age (±1 year) and parity (±1 delivery). Transvaginal ultrasound, magnetic resonance imaging or laparoscopy was used to diagnosed endometriosis, and the revised American Society for Reproductive Medicine score was used to classify the endometriosis into grade I/II versus grade III/IV. Male subfertility was defined in accordance with World Health Organization criteria (fifth edition). Results Compared with endometriosis cases, control couples with male subfertility had significantly higher rates of positive HCG test on day 16 (P = 0.047, OR 2.077, CI 1.009 to 4.276), ongoing implantation (defined as a positive fetal heart rate on transvaginal ultrasound at a gestational age of at least 6.5–7 weeks) (P = 0.038, OR 2.265, CI 1.048 to 4.893), ongoing pregnancy (defined by a vital pregnancy at 11 weeks) (P = 0.046, OR 2.292, CI 1.016 to 5.173) and live birth (P = 0.043, OR 2.502, CI 1.029 to 6.087). Conclusions After matching for embryo quality, woman's age and parity, rates of positive HCG tests, ongoing implantation, ongoing pregnancy and live birth were more than twice as high in the control group compared with the endometriosis group.

Research question Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? Design Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. Results Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. Conclusions Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.

Research question To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? Design The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification–warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. Results After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified–warmed oocytes (P 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. Conclusions Vitrified–warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified–warmed human oocytes is required.

Research question Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)? Design Evaluation of the spindle–chromosome complexes and intracellular distribution of inositol trisphosphate type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA. Assessment of the oocyte-related Ca2+ releasing capacity in response to Ca2+ ionophores and sperm microinjection in oocytes that failed to fertilize after ICSI or ICSI-AOA. Results IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar normalcy of spindle–chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups showed significant differences in proportion of normal or abnormal spindle–chromosome complex conformations. However, migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity to respond to stimuli such as exposure to Ca2+ ionophores or to sperm microinjection. Conclusions Evaluation of spindle–chromosome normalcy and distribution of IP3R1 does not help identify the presence of Ca2+ releasing deficiencies in these oocytes. However, oocyte Ca2+ analysis adds value in identifying Ca2+ releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca2+ machinery, which cannot be overcome by ICSI-AOA based on the use of Ca2+ ionophores.

Research question Does uterine activity differ in patients who have undergone successful IVF treatment compared with patients who have undergone unsuccessful IVF treatment? Design Prospective study of 16 women who underwent fresh single embryo transfer. All patients underwent transvaginal ultrasound in three phases of the IVF treatment: ovarian stimulation 1 h before embryo transfer (ET1) and 5–7 days after embryo transfer (ET5–7). Uterine motion analysis was implemented by a dedicated speckle tracking algorithm; frequency- and amplitude-related features were extracted from the derived signals to characterize the uterine activity in relation to ongoing implantation (positive HCG after 6 weeks) and ongoing pregnancy at 11 weeks. Results Uterine activity in terms of frequency (ovarian stimulation ET1, P = 0.04; ovarian stimulation ET5–7, P = 0.002) and amplitude (ovarian stimulation ET1, P = 0.0003; ovarian stimulation ET5–7, P = 0.000008) is significantly higher in the ovarian stimulation phase compared with ET1 and ET5–7. Women with ongoing pregnancies showed significantly higher uterine contraction frequency compared with those with no ongoing pregnancies in all phases (ovarian stimulation, P = 0.006; ET1, P = 0.015; ET5–7, P = 0.007). Uterine contraction amplitude was significantly lower (P = 0.037) in women at ET5–7 in women with ongoing pregnancies. Conclusions This study is a first step towards assessing uterine activity during IVF objectively and non-invasively. It is an essential step to understanding the previously suggested effect of contractions on IVF failure. Uterine activity after embryo transfer characterized by high frequency and low amplitude may favour embryo implantation. Research with larger patient cohorts is needed to build on current evidence and knowledge of uterine contractions during IVF.

Research question Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? Design This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P Results In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. Conclusions Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.

Introduction Heritable connective tissue disorders (HCTD) are a group of mostly autosomal dominant genetic diseases, characterized by cardiovascular involvement, tissue fragility, joint hypermobility and skin involvement. Some of them are associated with life threatening events such as aortic dissection and rupture, and a high mortality rate. We recently introduced Preimplantation Genetic Testing for monogenic disease (PGT-M) with a focus on HCTD and set up an explorative qualitative study to investigate the lived experiences and feelings of women, as well as their attitudes and psychological responses to PGT-M. Materials & Methods All couples are seen by a clinical geneticist and a psychologist, and blood samples of the couple are drawn for karyotyping, exclusion of carriership of cystic fibrosis, spinal muscular atrophy and fragile-X syndrome. Informative linked microsatellite markers flanking the causal (familial) mutation are identified for each couple. Once these parameters are defined, DNA, obtained from trophectoderm cells biopsied from a day 5-6 embryo, is genome-wide amplified. Subsequently, the informative linked microsatellite markers as well as the underlying disease causing mutation are analysed in each of these day 5-6 embryo's. In total, twelve HCTD couples have undergone the PGT-M procedure (FBN1: 4, SMAD3: 2, one each for TGFBR1, TGFB2, COL3A1, COL1A2, COL2A1 and WNT10A). In total, 52 embryos have been tested, 19 of them carried the healthy haplotype (microsatellite analysis) and did not carry the familial mutation (Sanger sequencing) and thus were suitable for transfer to the mother. Data of seven women, aged 26-39years, were collected by semi-structured interviews and interpreted by thematic analysis. At the time of the interviews, one participant had one child through PGT-M, two participants were pregnant after embryo transfer and four women did not have a successful embryo transfer leading to pregnancy yet. Results Our study shows that PGT-M is both physically and emotionally a demanding procedure. The main reason for choosing PGT-M is the severity of the disease. None of the couples wants to transmit the disease to their offspring. Since termination of pregnancy is not acceptable to them, PGT-M is preferred over prenatal diagnosis. The “drop-out race”, as all interviewed women call it, going from an encouraging number of available oocytes (after pick-up), to a limited number of useful embryos, is perceived as extremely stressful. Due to the frequent hospital visits and the prescribed strict timing of hormone injections, all women stated it is impossible to keep the PGT-M procedure hidden from their friends and colleagues. Women emphasize that receiving adequate information, during every step of the PGT-M process, is essential to learn more about the entire PGT-M trajectory. They also express the need of psychological support to help them in coping with this physical and psychological demanding technique. Conclusions PGT-M is a valuable option of reproductive technology for HCTD couples. Due to the significant emotional impact the PGT-M procedure causes, we advise that it should always go hand-in-hand with effective communication and psychological care in order to prevent distress in couples.

The aim of this qualitative study was to explore how recipients viewed payment of sperm donors. The study was conducted in Belgium, where, as in many countries, sperm donors receive recompense for their time and expenses. Face-to-face semi-structured interviews were conducted with 34 heterosexual and lesbian couples who, at the time of data collection, had at least one donor-conceived child aged 7–10 years or who were undergoing donor conception treatment. Although participants commonly described the issue of financial compensation as something that did not really concern them, all supported the idea that some level of payment was acceptable or even necessary. The participants also identified several ways in which donor payment offered advantages to their own position as (future) parents. Although the idea is commonly rehearsed that sperm donation is a gift and that monetary transaction for conception is demeaning, the participants of this study did not generally share this view. To them, a small financial return served as a symbolic acknowledgement of the donor's contribution and helped secure the type of relationship they expected from their donor. There was clearly concern, however, over high payments and the risk of attracting the wrong kind of donor.

Cross-border reproductive care (CBRC) is not a new concept, having been around since the beginning of assisted reproductive technology. Countries having taken the lead in developing new technologies have seen an influx of patients from other countries, because of legal limitations or the unavailability of good-quality care in their home country. This paper describes the experience of the Ghent University Hospital fertility centre with Dutch and French patients and tries to set out standards of care for CBRC patients. Dutch patients usually have longer histories, more complex pathology and are better informed, more outspoken and more financially secure. Thus, the care for these patients is challenging. The standards of care should be the same for local patients and CBRC patients; however, the nature of the complexity of the problems they come with will necessitate more time investment. Experience shows that many patients who have no access to treatment in their own country obtain reasonably good results. Some of them, however, are beyond possible help and these patients need a high standard of psychological care. All should be done to avoid that cross-border patients compromise the local care system. Special arrangements should be taken to manage possible complications following treatment. Cross-border reproductive care (CBRC) or the treatment of infertility in foreign patients, is not a new concept, having been around since the beginning of IVF technology. Countries having taken the lead in this field have seen an influx of patients from other countries, because of legal limitations or the unavailability of good quality care in their home country. This paper describes the experience of Ghent University Hospital fertility centre with Dutch and French patients and tries to set out standards of care for foreign patients. These patients usually have longer histories, more complex pathology and are better informed, more outspoken and more financially secure. Thus, the care for these patients is challenging. The standards of care should be the same for local and foreign patients; however, the nature of the complexity of the problems they come with will necessitate more time investment. Experience shows that many patients who have no access to treatment in their own country obtain reasonably good results. Some of them, however, are beyond possible help and these patients need a high standard of psychological care. All should be done to avoid that foreign patients compromise the local care system. Special arrangements should be taken to manage possible complications following treatment.

Interviews were conducted with patients undergoing treatment at the department for reproductive medicine at the University Hospital of Ghent, Belgium to describe how patients think about the use of embryos for science and how patients' views are related to their decision whether or not to donate their supernumerary embryos for science. Most participants knew little about the use of embryos for science. The perception of science and scientists, rather than the perception of the embryo, played an important role in the disposition decision making. The feeling of not having control over what would happen to their embryo and the fear that scientists would allow their embryo to develop into children were the main arguments against donation. This showed the importance of information about scientific research with embryos, such as the 14-day limit to keep embryos alive. Half of the participants pictured the medical team as the requesting party for their embryos and those who were not willing to donate indicated that they could be persuaded when asked directly by their caregivers. In conclusion, this study suggests that the perception of science, rather than the perception of the embryo, plays an important role in the decision to donate for science.

Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 μmol/l. An improved effect of a second exposure to 5 μmol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.

Isolation of human follicles is based on digestion of the tissue by combinations of enzymes. Follicle vitality and morphology are often based on the analysis of pooled follicles of different maturation stages. Information is therefore lacking on the effect of the isolation protocol to individual follicles of different maturation stages. A study was conducted using five protocols combining different enzymes and varying concentrations. Isolated follicles were classified according to their maturation stages, counted and characterized for vitality, morphology, early apoptosis and organization of transzonal projections. No statistical differences were found between the protocols when outcome parameters were analysed on a pool of follicles regardless of their maturation status. Differences were observed in quality when the follicles were analysed separately according to their maturation status. Combining morphologic characteristics and vitality, both Liberase DH and Liberase TM combined with collagenase IV were better at isolating high-quality primordial follicles, compared with collagenase IV. No statistical difference between the isolation protocols was found for primary follicles. If only high-quality isolated secondary follicles are needed, collagenase IV is found to be most advantageous. Follicles of different maturation stages react differently when enzymatic isolation protocols are compared.

A total of 3974 IVF and 1655 ICSI singleton births and 2901 IVF and 1102 ICSI twin births were evaluated. Pregnancies after both fresh and frozen transfers were included. IVF and ICSI singleton pregnancies were very similar for most obstetric and perinatal variables. The only significant difference was a higher risk for prematurity (

One of the most problematic issues of assisted reproduction is the high incidence of multiple pregnancies, resulting from the transfer of more than one embryo. Particularly at risk are young women who have good quality embryos. The only strategy to reduce the incidence of multiple pregnancies, including twin pregnancies, after assisted reproduction is single embryo transfer (SET). In 1997, the present authors therefore introduced elective SET (eSET) in this particular target group. The proportion of eSET increased from 1.5 (1997-1998) to 17.5% (1999-2002) of all transfers. In 2002, 20% of all transfers were SET. Comparing these two periods, an overall pregnancy rate of 35 and 34% per transfer, respectively, was obtained, while the overall twinning rate dropped from 30 to 21%. The twinning rate dropped to 14% in 2002, and in the eSET group there was only one monozygotic twin. These results demonstrate that a decline in the twinning rate is feasible without a drop in overall pregnancy rates. Comparing eSET with elective double embryo transfer (eDET), it was found that ongoing pregnancy and implantation rates were the same in both groups, but the proportion of twins was clearly different. It was further observed that the mean birthweight of singleton children born after eSET was significantly higher than that after DET. This could reflect a better developmental or implantation potential of these embryos, but this finding remains to be confirmed.

The potential role of embryo cryopreservation from the point of view of prevention of multiple pregnancies is analysed. Cryopreservation is an unavoidable option in stimulated IVF/intracytoplasmic sperm injection (ICSI), but at the same time an underestimated tool in the prevention of twins. There is a need for an evaluation system not only of the cryotechnology process per se , but also of the true augmenting effect of cryopreservation on the total reproductive potential of a single oocyte harvest. Only cryopregnancies occurring after an unsuccesful fresh cycle (possibly followed by one or more unsuccessful freeze–thaw cycles with embryos from the same harvest) truly reflect the augmentation potential of cryopreservation. This potential is greater than generally thought. First, the efficacy of cryopreservation is suboptimal with survival rates between 30 and 70%. Second, if single-embryo transfer were applied in a much larger proportion of cycles than is presently the case, more embryos would be available for cryopreservation, resulting in more and more successful freeze–thaw cycles. In the future, the combination of elective single-embryo transfer with an optimized cryopreservation programme is likely to become the standard of care for routine IVF/ICSI treatment.