The impact of histones linked to sperm chromatin on embryo development and ART outcome.

The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone-to-protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c-IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = -0.12, p < 0.05). Regarding the type of ART procedure (c-IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR<6%; p < 0.01) and 74.6% (HPR >26%; p < 0.01). The highest delivery rate (DR; 24.5%) was obtained for HPR within the range [6%; 26%]; DR was 21.9% for HPR<6% and 18.3% for HPR>26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy.

[Vitrification of oocytes and embryos: the law, the results, the future]. / Vitrification des embryons et des ovocytes: la loi, les résultats, les perspectives.

The French bio ethical law published in 2004 did not authorize the transfer of embryos submitted to a research program, or even issued from gametes concerned by an experimentation. Vitrification process was still considered as an "experimental" technique; thus it was impossible to vitrify either oocytes or embryos, whereas numerous international studies emphasized the interest of this technique for both oocytes and embryos, in particular if they were vitrified at the blastocyst stage. The new revised law (7/7/2011), clearly authorizes oocyte vitrification; moreover, studies intended to improve ART efficiency, are now permitted, enabling vitrification of embryos.

Methylation profile of the promoters of Nanog and Oct4 in ICSI human embryos.

STUDY QUESTION: What is the methylation status of the Nanog and Oct4 promoters in human gametes and ICSI embryos and is abnormal reprogramming of their methylation associated with developmental failure of ICSI embryos? SUMMARY ANSWER: Developmental failure of human ICSI embryos is associated with high methylation of the Oct4 promoter. WHAT IS KNOWN ALREADY: Nanog and Oct4 genes play critical roles in the establishment and maintenance of pluripotency during normal early embryonic development, and both are negatively regulated through the methylation of their promoters. STUDY DESIGN, SIZE AND DURATION: We analysed the methylation profile of Nanog and Oct4 promoters in 5 control sperm from normally fertile men, 70 metaphase II oocytes, 21 4-cell control ICSI embryos, 7 control blastocysts and 45 ICSI embryos arrested at 2- to 8-cell stage following prolonged culture. PARTICIPANTS, MATERIALS, SETTING AND METHODS: Embryos and gametes were donated for research by patients from the Department of Reproductive Medicine at the Hôpital Femme Mère Enfant (Bron, France) and the Clinique du Tonkin (Villeurbanne, France) after giving their informed consent. MAIN RESULTS: For both promoters, high methylation was observed in sperm cells. Although, in general, the promoters were unmethylated in oocytes, the methylation of some alleles was observed, particularly in oocytes from women with known infertility. Both gene promoters were hypomethylated in control blastocyst ICM (inner cell mass) and in control 2-8-cells embryos obtained from 6 out of 8 couples. However, they appeared highly methylated in embryos obtained from the other two couples. In most arrested ICSI embryos, the Nanog promoter was unmethylated while the Oct4 promoter was highly methylated. High methylation of the Oct4 promoter was significantly more pronounced in embryos from couples where a male factor was the only known cause of infertility. When the embryos were heterozygous for a G/A single nucleotide polymorphism, both alleles could be methylated, each likely representing a paternally inherited or a maternally inherited copy. LIMITATIONS AND REASONS FOR CAUTION: The study was done on a limited number of oocytes and embryos and the gametes of the couples were not available. WIDER IMPLICATIONS OF THE FINDINGS: These results provide new insight regarding the roles of epigenetic abnormalities in early developmental failure in humans. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. There was no competing interest.

Aneuploidy rate in spermatozoa of selected men with severe teratozoospermia.

The aim of this study was to evaluate the incidence of spermatic aneuploidies in men with severe teratozoospermia and to determine an eventual relation between aneuploidies and a specific morphology of spermatozoa. Fluorescence in situ hybridisation (FISH) using a probe cocktail containing the alpha satellite for the centromeric region of chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of thirty patients with severe teratozoospermia (abnormal forms >80%) and 15 fertile men with normal semen profiles. The mean frequency of teratozoospermia in patients was 91 ± 6.99%. There was statistically a significantly increased frequency of 1818, XY, XX and YY disomies in sperm with severe teratozoospermia compared with normal sperm (1.24% versus 0.08%, 1.42% versus 0.31%, 1.13% versus 0.19% and 1.11% versus 0.24%, respectively, P < 0.001 in all comparisons). The rate of total diploidy was significantly increased in patients compared with controls (1.46% versus 0.16%, P < 0.001). There was a correlation between macrocephalic spermatozoa and diploidy (r = 0.37, P < 0.05). Our data add further evidence that patients with severe teratozoospermia have an increased sperm aneuploidy rate and that this is particularly high in macrocephalic spermatozoa; FISH analysis on sperm could help to improve risk assessment and reproductive counselling in these individuals who are frequently candidates for intracytoplasmic sperm injection (ICSI) as a treatment of their infertility, as the use of ICSI has created consequential debate concerning the genetic risk for the offspring.

[The Bcl-2 family pathway in gametes and preimplantation embryos]. / Les régulateurs d'apoptose de la famille Bcl-2 dans les gamètes et lors du développement embryonnaire précoce.

Apoptosis, a form of cell death by self-destruction, has been reported in gametes and preimplantation embryos both in vitro and in vivo. Recent evidence suggests that cell death processes, whose control deserves to be elucidated, can impact embryo developmental competence. Moreover, quality of the gametes (particularly of the oocytes) is relevant not only for their survival rates but exert an influence during the early stages of embryo development. Thus, the investigation of apoptosis-related genes and mechanisms in early embryos is crucial. BCL-2 family proteins, through balanced interactions between pro- and anti-death members, play a pivotal role in controlling cell life and death. In this article, we review the literature concerning the expression of Bcl-2 family members in gametes and early embryos. Research results indicate that the various Bcl-2 subfamilies (pro- and anti-apoptotic "multidomain" family members and "BH3-only" death factors) exhibit a dynamic expression pattern during male and female gamete differentiation and early embryo development. While pro-apoptotic Bax protein plays a critical role in germ cell and early embryo degeneration, the relative importance of the prosurvival (Bcl-2, Bcl-xL, Bcl-w, Mcl-1) and "BH3-only" (Bim, Bad, Bik) members is not clear. Although information on expression patterns of Bcl-2 family transcripts and proteins is necessary, other elements such as transcriptional control (by environmental stimuli), subcellular localization and post-translational modifications should also be taken into account. Aside from basic research, a better understanding of apoptosis-related proteins and mechanisms involved in gamete and embryo viability at the molecular level may provide new guides for diagnosis and therapeutic strategies.

Prospective evaluation of the threat related to the use of seminal fractions from hepatitis C virus-infected men in assisted reproductive techniques.

BACKGROUND: The risk of hepatitis C virus (HCV) transmission during assisted reproductive techniques (ARTs) is still disputed and no report concerning its prospective evaluation is available. METHODS: The aim of this 4-year follow-up multicentre study that enrolled 86 HCV-serodiscordant couples was to determine whether a sperm-processing method was able to reduce levels of HCV in semen and the risk of HCV transmission to the newborn. All the men were chronically infected by HCV and 10 of them by human immunodeficiency virus. A total of 181 seminal plasmas and 153 sperm fractions were tested for the presence of HCV RNA. RESULTS: HCV RNA tested positive in 20.4% of the seminal samples. All of the 153 final sperm fractions tested negative for HCV. The detection of HCV RNA in semen was significantly correlated with a high viral load in blood (P < 0.05). The presence of HCV RNA in seminal plasma impaired neither semen parameters nor ART issue. From the 58 couples enrolled effectively in an ART programme, 24 pregnancies and 28 newborns were obtained. All of them tested negative for HCV RNA in blood. CONCLUSION: These results emphasize the safety of the semen-processing method. The negligible risk of transmitting HCV reduces the value of the systematic analysis of HCV RNA in seminal fractions prior to ART. Since use of this analytical procedure involves the freezing of semen, its avoidance would result in an increase in sperm quality and reduce the need to perform intracytoplasmic sperm injection techniques.

Dynamic CpG methylation of the KCNQ1OT1 gene during maturation of human oocytes.

BACKGROUND: Imprinted genes, many of which are involved in development, are marked during gametogenesis to allow their parent-of-origin specific expression, and DNA methylation at CpG islands is part of this epigenetic mark. Maternal imprint is apposed on oocyte during growth and maturation. Factors interfering with normal oocyte differentiation such as gonadotrophin stimulation and in vitro maturation (IVM) may possibly alter imprint resetting. METHODS: We examined the methylation of the KCNQ1OT1 differentially methylated region (KvDMR1) in human oocytes at different stages of their development: germinal vesicle (GV), metaphase I (MI) or metaphase II (MII). RESULTS: About 60% of alleles were fully methylated in GV oocytes and that full imprint is acquired in most MII oocytes. Similarly to in vivo, de novo methylation of DNA occurred in vitro during oocyte maturation. Following in vitro culture for 28 h, GV and MI oocytes are significantly more methylated when they are obtained from natural cycles than from patients undergoing gonadotrophin stimulation. CONCLUSION: This observation suggests that hyperstimulation likely recruits young follicles that are unable to acquire imprint at KvDMR1 during the course of the maturing process.

Specific hypermethylation of LINE-1 elements during abnormal overgrowth and differentiation of human placenta.

In human post-natal somatic cells, low global levels of DNA methylation have been associated with the hypomethylation of several repetitive elements, a feature that has been proposed to be a surrogate epigenetic marker. These data, mainly derived from the analysis of cancer cells, suggest a potential association between loss of cell-growth control and altered differentiation with hypomethylation of repetitive sequences. Partial hydatidiform moles (PHMs) can be used as an alternative model for investigating this association in a non-tumorigenic context. This gestational disease is characterized by abnormal overgrowth and differentiation of the placenta and spontaneous abortion. Here, we comprehensively analyse the DNA methylation of these trophoblastic tissues in both PHM and normal placenta at global and sequence-specific levels. Analysis of the global 5-methylcytosine content and immunohistochemistry indicate that PHM and normal placenta have identical global levels of DNA methylation. In contrast, bisulfite genomic sequencing shows that, whereas Alu, NBL2 and satellite 2 repetitive elements are equally methylated, LINE-1 sequences are hypermethylated in PHM tissues ( approximately 2-fold relative to normal placenta). Interestingly, altered demethylation is also found in triploid diandric embryos that originate from dispermic fertilization of an oocyte, a common event responsible for most PHMs. In conclusion, alterations of DNA methylation do not seem to be randomly distributed in PHM, as several repeated elements remain unaltered, whereas LINE-1 sequences are hypermethylated. In addition, our findings suggest that the hypomethylation of repetitive elements in cancer is directly linked to the neoplasic process and not a simple consequence of loss of growth control observed in most of the cancer cells.

[DNA sperm methylation in assisted reproductive techniques]. / Méthylation du spermatozoïde en Assistance médicale à la procréation (AMP).

In the last few years, many tests were developed to study the fertilizing properties of the spermatozoa. However none of them was useful to obtain a prognostic factor. Indeed, the integrity of the spermatic DNA is also necessary to a successful fertilization for obtaining a pregnancy. DNA integrity could be evaluated by the measurement of the level of DNA methylation. Indeed, in the mammals, the methylation of the ADN is involved in diverse processes amongst them the regulation of the genome expression during the embryonic development. The objective of this study is to evaluate the impact of the level of methylation of the spermatic DNA in the success of in vitro fertilization (IVF), in terms of rate of fertilization, quality of the embryos and rate of pregnancy. The immunostaining of the 5-methylecytosine, then the quantification by image analysis or with flow cytometry, allowed an objective evaluation of the level of total methylation of spermatic DNA. Our data show that the level of DNA methylation influences neither the fertilization rate nor the embryos quality. On the other hand, the rate of pregnancy is decreased if the total level of DNA methylation is lower than a threshold value. The level of spermatic DNA methylation represents a new parameter of spermatic maturation.

Analysis by fluorescence in situ hybridization (FISH) of the relationship between gonosomic aneuploidy and the results of assisted reproduction in men with severe oligozoospermia.

The aim of this study was to analyse the frequency of sex-chromosomal aneuploidy in human spermatozoa of severe oligozoospermic men undergoing intracytoplasmic sperm injection (ICSI), to evaluate the impact of these chromosomal anomalies on the results of the ICSI. Fluorescence in situ hybridization (FISH) with direct label fluorescence DNA probes specific for chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of 12 patients with severe oligozoospermia undergoing ICSI. A total of 500 spermatozoa were analysed per donor. The rate of gonosomal aneuploidy was significantly increased in the patients compared with normal donors (3.5% and 0.8% respectively). The sex-chromosomal anomalies due to meiosis I (XY) are less important than the anomalies caused by meiosis II (XX or XY), but the difference was not statistically significant. There was a negative correlation between the rate of aneuploidy and the percentage of spermatozoa with normal morphology (r = -0.71; P < 0.05). The correlation was negative between the percentage of gonosomic aneuploidy and the rate of fertilization (r = -0.7; P < 0.001). Our results suggest an increased rate of gonosomic aneuploidy in the patients with oligozoospermia compared with the normal population. This aneuploidy, although it decreases the rate of fertilization, does not seem to affect the rate of cleavage, nor the embryonic quality.

Geographic variations in probability of pregnancy in four cities of France.

BACKGROUND: Several studies have described geographic variations in human fecundability, but this phenomenon has almost exclusively been studied at an international level rather than within a given country. Our aim was to describe geographic variations in fecundability, the monthly probability of pregnancy, between four cities of France. METHODS: We conducted a cross-sectional study in four French maternity units from Toulouse, Rennes, Lyons and Paris, among partners of pregnant women. Women were asked about the time to pregnancy (TTP) of their current pregnancy. TTP was analysed with a discrete Cox model allowing to estimate fecundability ratios (FR). RESULTS: Time to pregnancy was defined for 894 couples. There was no strong evidence of heterogeneity in fecundability between the four compared cities (p=0.05 without adjustment and p=0.25 after adjustment for behavioural and medical factors). The highest fecundability was observed in Rennes and the lowest in Toulouse (fecundability ratio (FR)=1.28, 95% CI: 1.01-1.63). Differences in fecundability were smaller between the other cities. CONCLUSION: We highlighted a possibly slightly higher fecundability in Rennes compared to Toulouse. Possible explanations for this finding are discussed. We note that the finding is consistent with previous observations indicating a higher sperm concentration among semen donors in Rennes than in Toulouse.

[Testicular tissue cryoconservation for prepubertal boy: indications and feasibility]. / Cryoconservation de tissu testiculaire chez le garçon prépubère: indications et faisabilité.

Childhood cancers below 15 years old represent almost 2000 new cases each year; their evolution is often favourable since about 75% of children will recover from their cancer. However, the gonad, in particular the testis, is very sensitive to radiotherapy and alkylant agents. Cryopreservation of semen can be proposed to young adults before starting the treatment; the 14-17 years period can be considered as a critical phase, because some boys have not achieved puberty. Thereby semen collection failures are frequent and semen quality is sometimes poor. In prepubertal boys, testicular stem cell banking is the only way of preserving the future fertility. Transplantation of testicular tissue was tempted for the first time in 1994: recipient mice produced gametes derived from testicular stem cells of donor mice, and could produce transgenic offspring. However, in the mouse, births after transplantation of cryopreserved stem cells were obtained only recently, in 2003. A lot of questions remain uncompletely resolved in animal models and of course in humans: stem cells enrichment, freezing-thawing protocols, transplantation procedure and corresponding health risks, represented by the possible re-introduction of malignant cells. Xenotransplantation or in vitro maturation of spermatogonia after thawing the testicular tissue could represent an alternative permitting to obtain malignant cell-free spermatozoa. However, a complete spermatogenesis in vitro has not yet been obtained, in any species.

Influence of global sperm DNA methylation on IVF results.

BACKGROUND: In cases of male infertility, routine analysis for sperm characteristics is a poor predictive factor for the segmentation rate and embryo development in assisted reproductive technologies. It is assumed that epigenetic factors could have an influence on the embryo's quality. The aim of this work was to determine the relationship between sperm DNA methylation level and fertilization and pregnancy rates according to the assisted reproduction technique performed. METHODS: A prospective study was undertaken. Ejaculates were obtained from men (n = 63) undergoing an assisted reproduction procedure. 5-Methylcytosine was immunostained with a polyclonal antibody and revealed by fluorescein isothiocyanate. The DNA methylation level was then quantified by flow cytometry. RESULTS: Sixty-three conventional IVF cycles were performed, 760 oocytes were retrieved, an average of 8.1 +/- 4.8 embryos was obtained, and 2.4 embryos were transferred. Neither the fertilization rate nor the rate of good quality embryos was correlated with the DNA methylation level (r = -0.1 and r = -0.08 respectively; not significant). When sperm DNA methylation was >555 arbitrary units, the pregnancy rate was 33.3% compared with 8.3% in the lower (<555) group (P<0.05). CONCLUSION: DNA methylation level in human sperm could represent a new approach to study the ability of sperm to lead to pregnancy in an assisted reproduction procedure, especially when sperm samples with normal characteristics are used.

Selection bias in semen studies due to self-selection of volunteers.

BACKGROUND: Reports of a secular decrease in semen quality remain controversial, particularly due to the possibility of selection bias. We aimed to describe the potential bias due to self-selection of volunteers in semen studies involving fecund men. METHODS: Using data from the French multicentre study REPRHOM, we compared the characteristics of the partners of pregnant women for three levels of participation: completion of a refusal questionnaire (n = 698), agreement to complete the study questionnaires only (n = 676) and agreement to complete the study questionnaires and give a semen sample (n = 331, 13% of the subjects approached). RESULTS: Poorly educated men refused more often to participate than highly educated men. Semen providers were more likely to have experienced unfavourable pregnancy outcomes (odds ratio 1.68, 95% confidence interval 1.14-2.49) compared with participants completing the questionnaires only. Time to pregnancy was similar for all participants. CONCLUSIONS: This study demonstrates the existence of selection bias in semen studies associated with fertility and socio-demographic characteristics of men. The results of semen analysis for this population sample cannot be extrapolated to the whole population from which the volunteers originate. More information is required on who participates, and participation rates should be reported in semen studies to make it possible to interpret the results correctly.

[Assays for assessment of sperm DNA integrity: relationships with fertility and conceptus quality]. / Tests d'exploration de la qualité nucléaire du spermatozoïde: relations avec la fertilité et la qualité du conceptus.

Several assays are available for testing nuclear quality of spermatozoa, many of them allowing to define a DNA fragmentation index (DFI). Numerous recent studies on this subject agree on several points: negative correlations are observed between DFI and sperm characteristics. Concerning the relationships between DFI and artificial reproductive technologies, there are some disagreements about correlations between DFI and fertilization rates; conversely, in case of high DFI, both blastocyst formation rate and pregnancy rate are significantly reduced. Several authors have defined a threshold value for DFI, corresponding to an absence of pregnancy, or a very low pregnancy rate, for samples above this value. Unfortunately, there are no data available concerning the relationships between sperm DNA quality and abnormalities at birth.

Effects of cryopreservation on the meiotic spindle of human oocytes.

The microtubular meiotic spindle of most mammals, including humans, is very sensitive to cooling [Hum. Reprod. 16 (2001) 2374; Fertil. Steril. 54 (1990) 102; Fertil. Steril. 75 (2001) 769; Zygote 3 (1995) 357] and is rapidly depolymerised even after a slight reduction in temperature to 33 degrees C. Spindle disassembly is dependent on the extent of temperature decrease and its duration. After rewarming, the recovery is far from complete. Cryoprotectants themselves may alter the spindle structure, depending on the duration and temperature of exposure, the duration of recovery at 37 degrees C and the species [Hum. Reprod. Update 2 (1996) 193]. Damage to the meiotic spindle is considered to be the cause of aneuploid embryos, by inducing chromatid non-disjunction and chromosome scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Nevertheless, a consensus arose from all the studies: appropriate exposure to cryoprotectants and appropriate rates of cooling and thawing allow the cryopreservation of mature oocytes without any significant changes in their second meiotic spindle organisation and without any increase in the rate of aneuploid embryos [Mol. Hum. Reprod. 2 (1996) 445; Hum. Reprod. 8 (1993) 1101; Hum. Reprod. 9 (1994) 684; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 75 (2001) 354]. These fundamental studies in humans, showing good preservation of cell structures after freeze-thaw procedures opened the way to new successful clinical trials with embryos derived from cryopreserved mature oocytes [Fertil. Steril. 68 (1997) 724]. Considering immature oocyte freezing at prophase I (germinal vesicle (GV) stage), a stage which was thought to be less sensitive to cryoinjury, pooled data from the literature showed no advantage in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, conflicting results are reported on the effects of freezing on the spindle-chromosome configuration of immature oocytes or in vitro matured oocytes, highlighting the need for large scale studies [Hum. Reprod. 10 (1995) 1816; Hum. Reprod. 13 (Suppl. 3) (1998) 161; Hum. Reprod. 17 (2002) 1885; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 68 (1997) 920]. One child has been born after the use of cryopreserved immature oocytes at GV stage, matured in vitro and fertilised by ICSI [Hum. Reprod. 13 (1998) 3156], demonstrating at least the feasibility of this technique. Improvements are required so as to make mature and immature oocyte cryopreservation an established and safe technique for ART.

Pregnancy after safe IVF with hepatitis C virus RNA-positive sperm.

In France, assisted reproductive technology (ART) for hepatitis C virus (HCV)-infected patients is now subject to strict control after the publication of recent guidelines. Infertile serodiscordant couples (HCV-viraemic men and their seronegative female partners) require special care to carried out in designated 'viral risk' laboratories. Twelve sequential semen samples taken from an HCV chronically infected patient were analysed within 22 months. HCV RNA was detected in all the seminal plasma sampled before antiviral treatment with relatively high viral loads, and in two of the corresponding fractions of motile sperm obtained after a gradient selection, suggesting that a contamination risk by HCV through ART cannot be excluded. When the selection of sperm on a discontinuous gradient was followed by an additional swim-up step, HCV RNA was never detected in the motile sperm suspension that was frozen in highly secure straws. IVF was performed using cryopreserved sperm that tested negative for HCV RNA, resulting in a pregnancy. One month after embryo transfer, testing for HCV RNA and antibodies in the woman gave negative results.

Apoptosis in preimplantation mammalian embryo and genetics.

The presence of dead cells in the preimplantation mammalian embryo has been well described. Since Kerr et al. (1972), it has become apparent that these cells die by apoptosis, a form of programmed cell death. This review analyses the recent morphological and biochemical evidence that apoptosis play a role in early mammalian embryo development. Normal and apoptotic (i.e. fragmented) embryos express several apoptosis-related genes during mammalian preimplantation embryo development, with severe changes when apoptosis is activated; these findings support a model in which mammalian preimplantation embryo development is regulated by the ratio of pro- and -anti- apoptotic genes. Apoptosis may be a normal feature in human preimplantation development, even in vivo, and may play an active role in the developing embryo through the removal of genetically abnormal cells. Contrary to these beneficial effects, apoptosis may have detrimental effects if either the number of apoptotic cells or ratio of these cells to the normal cells is elevated. According to this value, embryos could either continue to develop or arrest.