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Nowadays, cryopreservation of gametes and embryos is a fundamental, integral, and indispensable part of infertility treatment or fertility preservation. Cryopreservation is not only needed for the policy of single embryo transfer and cryopreservation of surplus embryos, but for deferring embryo transfer in the case of ovarian hyperstimulation syndrome, uterine pathologies, and suboptimal endometrium built-up or when preimplantation genetic testing is needed. Several current strategies in assisted reproduction technology (ART) would be inconceivable without highly efficient cryopreservation protocols. Nevertheless, cryopreservation hampered for a long time, especially in terms of low survival rates after freezing and thawing. Only the technical progress during the last decades, namely, in regard to the implementation and advancement of vitrification, leveraged its application, and thus, even allows the cryopreservation of human oocytes—a process that is far from being easy. This review aims to provide a deeper insight into the physical processes of cryopreservation and to explore the character of the vitrified state in the extra and intracellular milieu in order to demonstrate that the common denominator to all cryopreservation procedures is the establishment of an intracellular amorphous condition that hinders the likelihood of crystallization.

What are the main risk factors associated with ectopic pregnancy and what is the true incidence of ectopic pregnancies in an IVF programme?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.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; P0.0001) 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; P0.0001). Transfer of slow-developing blastocysts (expressed by expansion) significantly increased the risk of ectopic pregnancy (OR 2.59; P = 0.0102).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.

Das „preimplantation genetic screening“ (PGS)/„preimplantation genetic testing for aneuploidies“ (PGT-A) ist ein Teil der Praimplantationsdiagnostik (PID). Der Begriff umfasst eine Vielzahl von Methoden, die angewandt werden, um die chromosomale Konstitution eines Embryos vor dessen Transfer in die Gebarmutter zu erfassen. Dabei geht es nicht etwa darum, die Weitergabe (mono‑)genetisch bedingter Erbkrankheiten zu verhindern, sondern rein um den Ausschluss von Aneuploidien. Mit der Anwendung eines PGS sollten die Implantationschancen sowie die Lebendgeburtenraten steigen und die Abortraten sinken, da euploide Embryonen fur den Transfer ausgewahlt werden, so die klinische Hypothese. In den letzten Jahren erfuhr diese Technik nicht nur eine rasante technische Entwicklung, sondern erfreut sich international auch einer zunehmenden Beliebtheit. Was dabei haufig nicht beachtet wird, ist die Tatsache, dass nach wie vor die klinische Evidenz fehlt, die den postulierten „benefit“ des PGS beweist. Selbst neueste Techniken, wie das „next generation sequencing“ (NGS), konnten bisher daran nichts andern. Im Gegenteil, diese hochsensitiven Analyseplattformen haben zu neuen Erkenntnissen uber den chromosomalen Status des Praimplantationsembryos gefuhrt und damit auch zu einem fundamentalen Problem, das das PGS allgemein infrage stellen konnte – namlich die Tatsache, dass ein nicht unwesentlicher Teil der menschlichen Embryonen nicht uniform euploid oder aneuploid ist, sondern als ein chromosomales Mosaik (CM) mit numerischen und/oder strukturellen Aberrationen vorliegt. Diese Ubersichtsarbeit fasst die aktuelle Literatur zusammen und durchleuchtet die Kernprobleme des PGS aus biologisch-medizinischer, juristischer und ethischer Sicht.

SummaryThe aim of this study was to investigate the effects of zona drilling and biopsy on day 3 followed by vitrification on day 5 on the cytoskeleton and development of human embryos, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy in human biopsied and non-biopsied embryos. In total, 98 human blastocysts (50 non-biopsied and 48 following biopsy on day 3) were vitrified on day 5 using either a commercial dimethyl sulphoxide (DMSO)-free vitrification kit or increasing concentrations of DMSO/EG (5%/5–10%/10–20%/20%). Following warming, the blastocysts were allowed to recover in culture for 24 h and were immunostained with α-tubulin, acetylated tubulin, and/or γ-tubulin antibodies in combination with 4′,6-diamidino-2-phenylindole (DAPI). Labelled embryos were examined by both fluorescence and confocal laser scanning microscopy. The survival rates following warming (92% non-biopsied vs 83.3% biopsied) and the incidence of normal spindle chromosome configurations was not statistically different between the two groups (65.2% non-biopsied vs 59.2% biopsied,P>0.05). The incidence of spindle abnormalities including multipolarity, chromosome lagging, congression failure and chromosome bridging were also similar between the two groups (P>0.05). This study is the first to compare the incidence of cytoskeletal abnormalities in biopsied and non-biopsied human embryos following vitrification. We conclude that there was no significant difference in the survival rates and the incidence of spindle abnormalities between the two groups.

STUDY QUESTION How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? SUMMARY ANSWER Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. WHAT IS KNOWN ALREADY Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. STUDY DESIGN, SIZE, DURATION This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. MAIN RESULTS AND THE ROLE OF CHANCE Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. LIMITATIONS AND REASONS FOR CAUTION Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. WIDER IMPLICATIONS OF THE FINDINGS Restriction of vitrification to top quality blastocysts may result in discarding potentially viable embryos. STUDY FUNDING AND COMPETING INTERESTS This study was not externally funded. There are no conflicts of interest to declare.

Intracytoplasmic sperm injection (ICSI) was introduced in the clinical practice in 1992, to overcome male-factor infertility and in failed conventional IVF cases. Today ICSI is used across the world in most IVF cases, for almost all indications of infertility and it provides consistently high pregnancy rates.

PURPOSE: The aim of this study was to compare the effect of the deselection of spermatozoa presenting vacuole-like structures using IMSI (intracytoplasmic morphologically selected sperm injection) with ICSI (intracytoplasmic sperm injection) by means of neonatal outcomes. METHODS: In a retrospective two-center analysis, a total of 848 successful IMSI or ICSI cycles ending with a live birth, induced abortion, or intrauterine fetal death (IUFD) were included. RESULTS: The IMSI and ICSI groups included 332 and 655 babies or fetuses, respectively. The parents were older in the IMSI group than in the ICSI group (mothers were 35.1 vs 32.9 years, and fathers were 39.1 vs 36.2 years). The multiple pregnancy rate was higher in the IMSI group. The mean pregnancy duration and mean birth weight were almost identical in both groups. There was no significant difference in major congenital malformations between the two groups. However, this rate was decreased in the IMSI group compared to that in the ICSI group (1.8 vs 3.2%), the difference being mainly found in singletons (1.4 vs 3.3%). Boys were more often affected than girls in both groups. The percentages of chromosomal abnormalities did not differ between the IMSI and ICSI groups (0.6 and 0.8%). The reported congenital malformations mainly affected the heart, urogenital, and musculoskeletal systems. CONCLUSIONS: In the present study, the malformation rates observed in the IMSI and ICSI groups were not significantly different, even if slightly lower after IMSI. However, the observed difference followed the same trends observed in previous reports, indicating the possible impact of IMSI on decreasing congenital malformation occurrences. This highlights the necessity to prospectively evaluate the impact of IMSI on neonatal outcome after IVF treatment.

Does the storage time of vitrified human blastocysts negatively impact their survival, the implantation potential of embryos or the malformation rate of babies born?There was no evidence that storage times of up to 6 years after vitrification (VIT) had a negative impact on blastocyst survival, the implantation potential of embryos or the malformation rate of babies born.Although several thousand children have been born after blastocyst VIT, many aspects of this technique remain to be elucidated. New applications, such as fertility preservation, lead to long storage times of vitrified gametes or embryos but it remains to be determined if these vitrified embryos are stable over time.A retrospective study including 603 transfers was conducted between January 2009 and April 2012. Blastocysts were vitrified using a closed system.All patients underwent the transfer of aseptically vitrified/warmed blastocysts in a cryo-cycle. A total of 1077 blastocysts were transferred. Survival rates (SRs), implantation potential, birth rates and characteristics of the children born were evaluated.We found that the storage of vitrified blastocysts in aseptic conditions neither impaired blastocyst viability (SR after warming during the first year of storage was 83.0% compared with 83.1% after 5-6 years of storage: NS) nor decreased pregnancy rates (clinical pregnancy rate after 1 year of storage was 40.0 versus 38.5% after 6 years: NS). In addition, no increase in the malformation rate over time was observed.Our study only included the transfer of blastocysts which had been vitrified aseptically (i.e. using a closed system). Therefore, our results might not be applicable to 'open' VIT systems. The long-term follow-up of children born will be necessary to confirm our findings.The results suggest that vitrified human blastocysts can be stored for long periods of time without significant negative consequences for the offspring. Therefore, the method should be of benefit to those patients who need to consider taking measures for fertility preservation.No external funding was sought for this study and the authors have no conflict of interest to declare.

Vitrification has been successfully applied in the cryopreservation of oocytes and embryos. It can be achieved either by direct (open system) or indirect (closed system) contact with liquid nitrogen. Unlike embryo vitrification, few reports have been published regarding oocyte vitrification in closed systems. In order to validate the effectiveness of a closed and aseptic vitrification approach for oocyte cryopreservation, a prospective, randomized study was performed. Sibling oocytes donated from the same donor were randomly and equally assigned into closed or open vitrification groups. A total of 75 vitrification-warming cycles were performed in each group. Apart from the survival rate (82.9% versus 91.0%, P

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification-warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency.

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.

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.

background: Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. methods: A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. results: In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P , 0.05). conclusions: The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.

The implementation of intracytoplasmic sperm injection (ICSI) in 1992 gave women whose male partners were diagnosed with severe male factor infertility the chance of having a baby using their partner’s own ejaculated [1] or testicular spermatozoa [2]. Despite reported fertilization rates of approximately 70%, fertilization is dramatically reduced in some cases and does not even occur in 1–3% of ICSI cycles. Most cases of fertilization failure following ICSI can be traced back to a lack of oocyte activation [3], while injection of ejaculated immotile sperm results in either poor or no fertilization [4, 5]. The reason for this lack of oocyte activation may be a deficient cytosolic sperm-associated oocyte activating factor (SAOAF) resulting in a partial or complete inability of the sperm to activate the oocyte [4] or to an inability of the oocyte to decondense the sperm [6]. In 1997, oocyte activation with calcium ionophore was tested in a clinical trial that reported the delivery of a healthy baby [7]. In the same year, oocyte activation with calcium ionophore was performed using injected spermatids [8]. There have been several reports of successful oocyte activation by calcium ionophore treatment in patients with repeated failure of fertilisation using ICSI [9–11] or after ICSI using round-headed spermatozoa [10].

Vitrification with the use of "Open" carrier devices (Cryoloop, cryotop, cryoleaf, Vitriplug) which allowed the contact with liquid nitrogen has become a more popular way to achieve cooling rate superior to 20,000 °C/min. Even though the question of contamination with liquid nitrogen during ultra-rapid cooling and storage remain debatable with the use of "open" devices, it is important to revise the carrier system in a way, which minimizes the risk of contamination. According to the EU tissues and cells directive, it is advisable that the cooling and storage should be carried out in embryo carrier devices ensuring complete separation of the embryos from liquid nitrogen in a way, which minimizes the risk of contamination. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier of aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. We developed an aseptic vitrification method of vitrification for MII oocytes and embryos at different stage of development using the "VitriSafe" as "closed" carrier device.

The use of high levels of cryoprotectants (CPs) in solutions applied to vitrify oocytes or embryos is an argument to still prefer slow freezing procedure. Is it a justified argument? Out of three studies using mice zygotes we may assume that (i) the intracellular concentration of CPs is far lower than the one in the vitrification solutions, (ii) the intracellular concentration of CPs in the vitrified zygote is in contrary to the common beliefs even lower than the one observed after a slow freezing procedure, (iii) survival after slow freezing reflects the presence of an intracellular vitrified state in these cells.

Although some post-thaw morphological predictors of pregnancy have been investigated in slow freezing of blastocysts, no such data have been published for vitrified and warmed blastocysts. Therefore, a prospective four-part score was applied to vitrified/warmed day-5 embryos to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy and live birth. All morulae/blastocysts that were considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion, hatching (out of an artificial gap in the zona pellucida), extensive cytoplasmic granulation and presence of necrotic foci. Overall, 74% (202/273) of the vitrified concepti were found to be viable after warming. Early blastocysts showed better survival versus extended/hatching blastocysts (P < 0.01). Of the morphological parameters analysed, immediate re-expansion (P < 0.05) and hatching (P < 0.001) were positive predictors of the rates of implantation, pregnancy and live birth. The opposite holds for extensive cytoplasmic granulation (P < 0.05), which was negatively related. Accurate scoring of warmed blastocysts (within the first 2 h) allows for prediction of pregnancy outcome, and thus will help to further reduce the number of transferred embryos.

Objective To evaluate whether is possible to vitrify oocytes in an aseptic (hermetically closed) fashion and maintain clinical results comparable with those of fresh oocytes. Design Prospective, observational, cohort, noninferiority trial. Setting Private in vitro fertilization center. Patient(s) One hundred eighty-four recipients of donated vitrified oocytes. Intervention(s) Closed system vitrification. Main Outcome Measure(s) Pregnancy rate per cycle and clinical pregnancy rate per cycle. Result(s) No statistically significant differences were observed between two groups regarding the pregnancy rate per cycle (63.1% vs. 60.9%) or the clinical pregnancy rate per cycle (55.4% vs. 58.7%). Biochemical pregnancy rate was statistically significantly higher in the fresh group (7.6% vs. 2.2%). The mean number of embryos transferred was similar (2.0 ± 0.0 vs. 1.97 ± 0.3). Concerning embryologic data, there were no statistically significant differences regarding the fertilization, cleavage, top quality day-3 embryo, or blastocyst rates, whereas the top quality blastocyst rate on day 5 was statistically significantly higher in the fresh oocyte group (31.7% vs. 26.1%). Conclusion(s) Aseptically (in a closed system) vitrified oocytes show similar clinical efficiency compared with their sibling fresh oocytes.

INTRODUCTION Vitrication is now a part of nearly every in vitro fertilization (IVF) cycle. Gametes and embryos are, therefore, stored in liquid nitrogen (LN2) for a number of years and possibly decades, especially when considering donor cryo-banks and gamete freezing for social reasons or fertility preservation. Many cryopreserved samples within IVF are stored in containers that are not hermetically sealed, which is not in concordance with medical directives for the storage of biological tissues. Such directives have been put in place to minimize the risk of cross-contamination from infected material and also the LN2 itself, which is not sterile.

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. 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. A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P