Embryo drop-out rates in preimplantation genetic testing for aneuploidy (PGT-A): a retrospective data analysis from the DoLoRes study.

PURPOSE: To evaluate the decline in transferable embryos in preimplantation genetic testing for aneuploidy (PGT-A) cycles due to (a) non-biopsable blastocyst quality, (b) failure of genetic analysis, (c) diagnosis of uniform numerical or structural chromosomal aberrations, and/or (d) chromosomal aberrations in mosaic constitution. METHODS: This retrospective multicenter study comprised outcomes of 1562 blastocysts originating from 363 controlled ovarian stimulation cycles, respectively, 226 IVF couples in the period between January 2016 and December 2018. Inclusion criteria were PGT-A cycles with trophectoderm biopsy (TB) and next generation sequencing (NGS). RESULTS: Out of 1562 blastocysts, 25.8% were lost due to non-biopsable and/or non-freezable embryo quality. In 10.3% of all biopsied blastocysts, genetic analysis failed. After exclusion of embryos with uniform or chromosomal aberrations in mosaic, only 18.1% of those originally yielded remained as diagnosed euploid embryos suitable for transfer. This translates into 50.4% of patients and 57.6% of stimulated cycles with no euploid embryo left for transfer. The risk that no transfer can take place rose significantly with a lower number of oocytes and with increasing maternal age. The chance for at least one euploid blastocyst/cycle in advanced maternal age (AMA)-patients was 33.3% compared to 52.1% in recurrent miscarriage (RM), 59.8% in recurrent implantation failure (RIF), and 60.0% in severe male factor (SMF). CONCLUSIONS: The present study demonstrates that PGT-A is accompanied by high embryo drop-out rates. IVF-practitioners should be aware that their patients run a high risk of ending up without any embryo suitable for transfer after (several) stimulation cycles, especially in AMA patients. Patients should be informed in detail about the frequency of inconclusive or mosaic results, with the associated risk of not having an euploid embryo available for transfer after PGT-A, as well as the high cost involved in this type of testing.

Suboptimal endometrial-embryonal synchronization is a risk factor for ectopic pregnancy in assisted reproduction techniques.

RESEARCH QUESTION: What are the main risk factors associated with ectopic pregnancy and what is the true incidence of ectopic pregnancies in an IVF programme? DESIGN: Retrospective single-centre study of 12,429 blastocyst transfers (8182 fresh and 4247 frozen embryo transfers) conducted between January 2010 and December 2017. IVF outcome was analysed, and ectopic pregnancy risk evaluated according to patient's characteristics and assisted reproductive technology treatment factors. RESULTS: Of 5061 patients reporting a positive pregnancy test, 43 were diagnosed with ectopic pregnancy (0.85%). Neither female age (36.7 versus 35.8 years), body mass index, quality of transfer nor stimulation protocol affected the ectopic pregnancy rate, but history of previous ectopic pregnancy (OR 3.26; P = 0.0080), tubal surgery, or both (OR 6.20; P < 0.0001) did. The incidence of ectopic pregnancy was increased in women with uterine malformations (OR 3.85; P = 0.0052), uterine pathologies (OR 5.35; P = 0.0001), uterine surgeries (OR 2.29; P = 0.0154) or sub-optimal endometrial build-up (OR 4.46 to 5.31; P < 0.0001). Transfer of slow-developing blastocysts (expressed by expansion) significantly increased the risk of ectopic pregnancy (OR 2.59; P = 0.0102). CONCLUSIONS: Unfavourable uterine environment, including uterine pathologies, uterine or tubal surgery and suboptimal endometrial build-up were related to ectopic pregnancy. Low expansion grade of blastocysts was identified as an additional putative risk factor for ectopic pregnancy, indicating the importance of proper embryonal-maternal synchronization. The overall ectopic pregnancy rate after blastocyst transfer was low, comparable with reported ectopic pregnancy rates in spontaneous conceptions. Proper evaluation of tubal and uterine pathologies, optimizing endometrial preparation and the transfer of expanded blastocysts in a frozen embryo transfer cycle, might be beneficial.

The impact of paternal factors on cleavage stage and blastocyst development analyzed by time-lapse imaging-a retrospective observational study.

PURPOSE: Various time-lapse studies have postulated embryo selection criteria based on early morphokinetic markers. However, late paternal effects are mostly not visible before embryonic genome activation. The primary objective of this retrospective study was to investigate whether those early morphokinetic algorithms investigated by time-lapse imaging are reliable enough to allow for the accurate selection of those embryos that develop into blastocysts, while of course taking into account the correlation with the type of injected spermatozoa. METHODS: During a period of 18 months, a total of 461 MII oocytes from 43 couples with severe male factor infertility and previous "external" IVF failures after cleavage-stage embryo transfer (ET) were fertilized by intracytoplasmic morphologically selected sperm injection (IMSI). Thereof, 373 embryos were monitored in a time-lapse incubator until ET on day 5. Blastocyst outcome in combination with three previously postulated MKc (cc2: t3-t2, 5-12 h; t3, 35-40 h; t5, 48-56 h) and the morphology of the selected sperm were analyzed. RESULTS: A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P < 0.01) and top blastocysts (25.3 vs. 10.8 %; P < 0.001) was observed in the group of those meeting all three morphokinetic criteria (MKc3). However, MKc3 were only met in 23.3 % of all embryos. Moreover, TBR was influenced by the type of injected spermatozoa. In both groups, TBR decreased dramatically (MKc3, 35.0 vs. 17.0 %; MKc < 3, 14.2 vs. 8.4 %) when class II/III sperm instead of class I were injected. CONCLUSION: Early morphokinetic parameters might give some predictive information but fail to serve as a feasible selective tool for the prediction of blastocyst development given the influence of the type of spermatozoa injected.

Intrauterine administration of human chorionic gonadotropin does not improve pregnancy and life birth rates independently of blastocyst quality: a randomised prospective study.

BACKGROUND: Successful embryo implantation depends on a well-timed maternal-embryonic crosstalk. Human chorionic gonadotropin (hCG) secreted by the embryo is known to play a key role in this process and to trigger a complex signal transduction cascade allowing the apposition, attachment, and invasion of the embryo into the decidualized uterus. Production of hCG was reported to be dependent on blastocyst quality and several articles suggested that intrauterine hCG injection increases pregnancy and implantation rates in IVF patients. However, no study has as yet analysed birth rates as final outcome. Our objective was to determine whether clinical outcome after blastocyst transfer can be improved by intrauterine injection of hCG and whether this is dependent on blastocyst quality. METHODS: A prospective randomised study was conducted in two settings. In cohort A, hCG application was performed two days before blastocyst transfer. In cohort B, the administration of hCG occurred just prior to embryo transfer on day 5. For both cohorts, patients were randomised to either intrauterine hCG application or to the control group that received culture medium. Clinical outcome was analysed according to blastocyst quality of transferred embryos. RESULTS: The outcome of 182 IVF-cycles (cohort A) and 1004 IVF-cycles (cohort B) was analysed. All patients received a fresh autologous blastocyst transfer on day five. Primary outcomes were pregnancy rates (PR), clinical pregnancy rates (cPR), miscarriage rates (MR), and live birth rates (LBR). No improvement of clinical outcome after intrauterine hCG administration on day 3 (cohort A) or day 5 (cohort B) was found, independently of blastocyst quality transferred. The final outcome in cohort A: LBR after transfer of top blastocysts was 50.0 % with hCG and 53.3 % in the control group. With non-top blastocysts, LBR of 17.1 % (hCG) and 18.2 % (control) were observed (n.s.). In cohort B, LBR with top blastocysts was 53.3 % (hCG) and 48.4 % (control), with non-top blastocysts it came to 28.7 % (hCG) and 35.0 % (control). The differences between the groups were statistically not significant. Furthermore, we investigated a possible benefit of hCG administration in correlation with female age. In both age groups (<38 years and ≥ 38 years) we found similar LBR after treatment with hCG vs. medium. A LBR of 47.1 % vs. 48.7 % was obtained in the younger group and 26.6 % vs. 30.8 % in the older group. CONCLUSIONS: In contrast to previous studies indicating a substantial benefit from intrauterine hCG application in cleavage stage embryo transfers, in our study we could not find any evidence for improvement of clinical outcome in blastocyst transfer cycles, neither with top nor with non-top quality morphology.

Transfer of blastocysts with deviant morphological and morphokinetic parameters at early stages of in-vitro development: a case series.

Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage.

Sperm head vacuoles are not affected by in-vitro conditions, as analysed by a system of sperm-microcapture channels.

Since the introduction of the motile sperm organelle morphology examination, there has been increasing recognition of the fact that the presence of large nuclear vacuoles might have deleterious effects on embryo development. Nevertheless, one fundamental question still being debated is whether specific in-vitro conditions during the handling of semen have an impact on vacuole formation. This study's objective was to analyse whether incubation temperature (20, 37°C) or oxidative stress stimulates the formation of nuclear vacuoles. Furthermore, it examined whether vacuoles disappear in the presence of an acrosome reaction inducer. Therefore, a system of sperm-microcapture channels was developed to permit the observation of the same living spermatozoa over a period of 24h. Neither incubation at 37°C nor induction of oxidative stress led to de-novo formation of nuclear vacuoles. Induction of the acrosome reaction using calcium ionophore A23587 did not lead to any modifications in the proportion of spermatozoa with vacuoles or to the disappearance of pre-existing vacuoles. According to these observations, it is concluded that nuclear vacuoles on the sperm head are already produced at earlier stages of sperm maturation and are not induced or modulated by routine laboratory environments. The examination of spermatozoa at very high magnification has led to the increasingly widespread recognition that the presence of large vacuoles in the human sperm head has deleterious effects on embryo development. One fundamental question, however, still remains: do specific conditions in the laboratory during the preparation and the handling of semen have an impact on vacuole formation? Our initial objective was to analyse whether different incubation temperatures (20, 37°C) and the induction of oxidative stress lead to the formation of sperm head vacuoles. Furthermore, we examined whether vacuoles disappear in the presence of an acrosome reaction inducer. In order to do this we developed a system of sperm-microcapture channels, which permits the observation of the same living spermatozoa over a period of 24h. Incubation at 37°C or induction of oxidative stress did not lead to the formation of any new vacuoles. After inducing the acrosome reaction, we did not detect any modification in the proportion of vacuolated spermatozoa. According to our observations, different temperatures or environmental conditions in the laboratory have no impact on the formation or disappearance of vacuoles. We conclude that sperm head vacuoles are already produced at earlier stages of sperm maturation.

The combination matters--distinct impact of lifestyle factors on sperm quality: a study on semen analysis of 1683 patients according to MSOME criteria.

BACKGROUND: Poor sperm quality can negatively affect embryonic development and IVF outcome. This study is aimed at investigating the influence of various lifestyle factors on semen quality according to MSOME (motile sperm organelle morphology examination) criteria. METHODS: 1683 male patients undergoing assisted reproductive technologies (ART) in our clinic were surveyed about their age, BMI (body mass index), ejaculation frequency, nutrition, sports, sleeping habits and social behavior. Semen samples were collected and evaluation of semen parameters according to MSOME and WHO criteria was performed. Results were grouped and statistically analyzed. RESULTS: Although single parameters had minor effects on sperm parameter, the combination of age, BMI, coffee intake, ejaculatory frequency and duration of sexual abstinence were identified as factors having a negative effect on sperm motility. Additionally, we could demonstrate that MSOME quality was reduced. The negative impact of age, BMI and coffee intake on sperm quality could be compensated if patients had a high ejaculation frequency and shorter periods of sexual abstinence. CONCLUSIONS: Combinations of adverse lifestyle factors could have a detrimental impact on sperm, not only in terms of motility and sperm count but also in terms of sperm head vacuolization. This negative impact was shown to be compensated by higher ejaculation frequency and a shorter period of sexual abstinence. The compensation is most likely due to a shorter storage time in the male gonads, thus reducing the duration of sperms' exposure to reactive oxygen species (ROS).

Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment.

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle. In stimulated IVF cycles, high doses of hormones are given to stimulate multifollicular growth. One drawback of the hormonal substitution is that the uterine environment is not at the same time optimally prepared for embryo implantation. A solution, which is increasingly under discussion, is to cryopreserve the embryos obtained in the stimulated cycle and to transfer them back into the optimal uterine environment in a subsequent cryo-cycle. This procedure requires highly secure and safe cryopreservation protocols in order to ensure benefits for both pregnancy and birth rates. We have established a protocol for the vitrification of zygote-stage embryos in aseptic devices, which minimize the potential risk of contamination during cooling and storage. The vitrified zygotes showed the same blastocyst development as compared with sibling zygotes in fresh culture. A clinical study comprising 173 cryo-cycles with transfer of blastocysts originating from vitrified zygotes shows an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification conditions contributes to a change in transfer strategy and encourages us to increase the cryo-embryo transfer rate for an optimal uterine environment.

Dietary supplementation of antioxidants improves semen quality of IVF patients in terms of motility, sperm count, and nuclear vacuolization.

BACKGROUND: This study aimed to investigate the influence of an oral antioxidative supplementation on sperm quality of in vitro fertilization (IVF) patients, as analyzed by sperm motility according to the WHO criteria and motile sperm organelle morphology examination (MSOME). METHODS: Semen samples were collected from 147 patients before undergoing an IVF/intracytoplasmic morphologically-selected sperm injection (IMSI) cycle and 2 - 12 months after an antioxidative supplementation. Semen analysis was evaluated according to WHO and MSOME criteria. Spermatozoa were grouped according to the size of nuclear vacuoles within the sperm's heads. Patients were divided into oligoasthenoteratozoospermic (OAT) and non-OAT men. Between first and second semen analysis, patients were supplemented orally with an antioxidative preparation. RESULTS: After the antioxidative therapy we observed a significant reduction in the percentage of immotile sperm cells in the patients. Additionally, the percentage of class I spermatozoa according to MSOME criteria was significantly higher after antioxidative supplementation. In OAT patients the percentage of class I sperm was found to be increased, although not significantly. However, we observed a drastic improvement in sperm motility as well as in total sperm count in this group. CONCLUSION: The results demonstrated a considerable improvement in semen quality, notably in OAT patients. Considering the putative relationship between semen quality on the one hand and reactive oxygen species on the other, the observed changes in the sperm parameters indicate that a decline in semen quality, and even subtle morphological changes, might be associated with oxidative stress. Our findings suggest that an antioxidative and micronutrient supplementation has a remarkable benefit for IVF patients having restricted sperm parameters, in particular.

Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.

Spermatozoa selection at high magnification before intracytoplasmic sperm injection seems to be positively associated with pregnancy rates after day 3 embryo transfers. The aim was to demonstrate an association between the presence of vacuoles in sperm nuclei and the competence of embryos to develop to day 5. Grading of spermatozoa at x 6000-x 12,500 magnification: grade I, no vacuoles; grade II, or=1 large vacuole; grade IV, large vacuoles with other abnormalities. The outcome of embryo development in a group of 25 patients after sibling oocyte injection with the four different grades of spermatozoa showed no significant difference in embryo quality up to day 3. However, the occurrence of blastocyst formation was 56.3 and 61.4% with grade I and II spermatozoa respectively, compared with 5.1% with grade III and 0% with grade IV respectively (P < 0.001). Spermatozoa selection at high magnification using Nomarski interference contrast is useful to identify more precisely the size and the number of nuclear vacuoles that greatly exert a negative effect on embryo development to the blastocyst stage. These observations confirm previous studies pointing to possible 'early and late paternal effects', both of which may have an impact on early embryonic development.