Biological age of the endometrium using DNA methylation.

Age has a detrimental effect on reproduction and as an increasing number of women postpone motherhood, it is imperative to assess biological age in terms of fertility prognosis and optimizing fertility treatment individually. Horvath's epigenetic clock is a mathematical algorithm that calculates the biological age of human cells, tissues or organs based on DNA methylation levels. The clock, however, was previously shown to be highly inaccurate for the human endometrium, most likely because of the hormonal responsive nature of this tissue. The aim of this study was to determine if epigenetically based biological age of the human endometrium correlated with chronological age, when strictly timed to the same time point in the menstrual cycle. Endometrial biopsies from nine women were obtained in two consecutive cycles, both strictly timed to the LH surge (LH + 7) and additionally, peripheral whole blood samples were analyzed. Using the Illumina HumanMethylation 450 K array and Horvath's epigenetic clock, we found a significant correlation between the biological age of the endometrium and the chronological age of the participants, although the endometrial biological age was accelerated by comparison with blood and chronological age. Moreover, similar biological ages were found in pairs of consecutive biopsies, indicating that an endometrial biopsy does not alter the biological age in the following cycle. In conclusion, as long as endometrial samples are timed to the same time point in the menstrual cycle, Horvath's epigenetic clock could be a powerful new biomarker of reproductive aging in the human endometrium.

Proposed guidelines on the nomenclature and annotation of dynamic human embryo monitoring by a time-lapse user group.

STUDY QUESTION: Can the approach to, and terminology for, time-lapse monitoring of preimplantation embryo development be uniformly defined in order to improve the utilization and impact of this novel technology? SUMMARY ANSWER: The adoption of the proposed guidelines for defining annotation practice and universal nomenclature would help unify time-lapse monitoring practice, allow validation of published embryo selection algorithms and facilitate progress in this field. WHAT IS KNOWN ALREADY: An increasing quantity of publications and communications relating to time-lapse imaging of in vitro embryo development have demonstrated the added clinical value of morphokinetic data for embryo selection. Several articles have identified similar embryo selection or de-selection variables but have termed them differently. An evidence-based consensus document exists for static embryo grading and selection but, to date, no such reference document is available for time-lapse methodology or dynamic embryo grading and selection. STUDY DESIGN, SIZE AND DURATION: A series of meetings were held between September 2011 and May 2014 involving time-lapse users from seven different European centres. The group reached consensus on commonly identified and novel time-lapse variables. PARTICIPANTS/MATERIALS, SETTING, METHODS: Definitions, calculated variables and additional annotations for the dynamic monitoring of human preimplantation development were all documented. MAIN RESULTS AND THE ROLE OF CHANCE: Guidelines are proposed for a standard methodology and terminology for the of use time-lapse monitoring of preimplantation embryo development. LIMITATIONS, REASONS FOR CAUTION: The time-lapse variables considered by this group may not be exhaustive. This is a relatively new clinical technology and it is likely that new variables will be introduced in time, requiring revised guidelines. A different group of users from those participating in this process may have yielded subtly different terms or definitions for some of the morphokinetic variables discussed. Due to the technical processes involved in time-lapse monitoring, and acquisition of images at varied intervals through limited focal planes, this technology does not currently allow continuous monitoring such that the entire process of preimplantation embryo development may be visualized. WIDER IMPLICATIONS: This is the first time that a group of experienced time-lapse users has systematically evaluated current evidence and theoretical aspects of morphokinetic monitoring to propose guidelines for a standard methodology and terminology of its use and study, and its clinical application in IVF. The adoption of a more uniform approach to the terminology and definitions of morphokinetic variables within this developing field of clinical embryology would allow practitioners to benefit from improved interpretation of data and the sharing of best practice and experience, which could impact positively and more swiftly on patient treatment outcome. STUDY FUNDING/COMPETING INTERESTS: There was no specific funding for the preparation of these proposed guidelines. Meetings were held opportunistically during scientific conferences and using online communication tools. H.N.C. is a scientific consultant for ESCO, supplier of Miri TL. I.E.A. is a minor shareholder in Unisense Fertilitech, supplier of the EmbryoScope. Full disclosures of all participants are presented herein. The remaining authors have no conflict of interest.

Limitations of a time-lapse blastocyst prediction model: a large multicentre outcome analysis.

The goal of embryo selection models is to select embryos with the highest reproductive potential, whilst minimizing the rejection of viable embryos. Ultimately, any embryo selection model must be tested on clinical outcome. We therefore retrospectively tested a published blastocyst prediction model on a large combined set of transferred embryos with known clinical outcome. The model was somewhat effective in that we found a relative increase of 30% for implantation in the model-selected group of embryos. There was, however, a concomitant large rejection of embryos from our test cohort, which actually resulted in pregnancy. This hypothetical experiment highlights the limitations of predicting blastulation only. Crucially, it illustrates that both sensitivity and specificity are important parameters when developing embryo selection models for prospective clinical use.

Triploid pregnancies: genetic and clinical features of 158 cases.

OBJECTIVE: The purpose of this study was to analyze the correlation between the genetic constitution and the phenotype in triploid pregnancies. STUDY DESIGN: One hundred fifty-eight triploid pregnancies were identified in hospitals in Western Denmark from April 1986 to April 2010. Clinical data and karyotypes were collected retrospectively, and archived samples were retrieved. The parental origin of the genome, either double paternal contribution (PPM) or double maternal contribution (MMP) was determined by an analysis of methylation levels at imprinted sites. RESULTS: There were significantly more PPM than MMP cases (P < .01). In MMP cases, the possible karyotypes had similar frequencies, whereas, in PPM cases, 43% had the karyotype 69,XXX, 51% had the karyotype 69,XXY, and 6% had the karyotype 69,XYY. Molar phenotype was seen only in PPM cases. However, PPM cases with a nonmolar phenotype were also seen. For both parental genotypes, various fetal phenotypes were seen at autopsy. Levels of human chorionic gonadotropin in maternal serum were low in MMP cases and varying in PPM cases, some being as low as in the MMP cases. CONCLUSION: In a triploid pregnancy, suspicion of hydatidiform mole at ultrasound scanning, by macroscopic inspection of the evacuated tissue, at histology, or because of a high human chorionic gonadotropin in maternal serum level each predict the parental type PPM with a very high specificity. In contrast, the sensitivity of these observations was <100%.

Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method: a time-lapse study.

PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p < 0.001), displayed longer duration of the 3-cell stage (p < 0.001), and arrested development from the compaction stage and onwards (p < 0.001). For the IVF embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0.001). More 3PN IVF than ICSI embryos displayed early cleavage into 3 cells (p < 0.001). CONCLUSIONS: This study reports differences in cleavage patterns between 2PN and 3PN embryos and for the first time demonstrates differences in the cleavage pattern between 3PN IVF and ICSI embryos.

Time-lapse monitoring as a tool for clinical embryo assessment.

As elective transfer of a single embryo (eSET) becomes increasingly accepted, the need to improve implantation rates becomes crucial. Selecting the most competent embryo therefore constitutes a major challenge in assisted reproductive technology. Embryo morphology and developmental stage at given time points are closely correlated with developmental competence and assessment of morphological parameters at discrete inspection points thus remains the preferred way of evaluating embryonic potential. Lately, more attention has been given to the assessment of dynamic embryo development as a tool for evaluating embryonic potential. The introduction of time-lapse equipment approved for use on human embryos offers novel clinical opportunities for continuous monitoring of embryos, enabling flexible evaluation of known morphological parameters and potentially introducing new dynamic markers of viability. Due to lack of larger, randomized clinical studies it remains to be elucidated whether embryo selection using dynamic parameters improves clinical outcome and which parameters are of significance. Before such randomized controlled studies are organized, the most promising parameters to evaluate must be identified. This mini-review summarizes the current knowledge about dynamic markers of viability and discusses the potential clinical role of time-lapse analysis in embryo assessment and selection.

Microselection--affinity selecting antibodies against a single rare cell in a heterogeneous population.

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.

Culture of human oocytes with granulocyte-macrophage colony-stimulating factor has no effect on embryonic chromosomal constitution.

The effect on ploidy rate in donated human oocytes after in-vitro culture with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; 2 ng/ml) from fertilization until day 3 was examined in a multicentre, prospective placebo-controlled and double-blinded study including 73 women donating 86 oocytes. The primary endpoint was to investigate the chromosomal constitution of human embryos (fluorescence in-situ hybridization analysis for chromosomes 13, 16, 18, 21, 22, X and Y) cultured with or without GM-CSF. The secondary endpoints were number of top-quality embryos (TQE) and number of normally developed embryos evaluated morphologically on day 3. The cytogenetic analyses demonstrated non-inferiority and therefore the chromosomal constitution of human embryos cultured in vitro in the presence of 2 ng/ml GM-CSF was no worse than the control group cultured without GM-CSF. In-vitro culture of human embryos in the presence of 2 ng/ml GM-CSF resulted in 34.8% (8/23) uniformly normal embryos. Culture without 2 ng/ml GM-CSF resulted in 33.3% (9/27) uniformly normal embryos. A trend towards a higher number of TQE in the test group was observed; however, due to lack of TQE in the control group, this was considered a random finding.

Diagnostic Accuracy of a Point-of-Care Test for Celiac Disease Antibody Screening among Infertile Patients.

BACKGROUND: Screening for celiac disease among infertile patients has been suggested. Several rapid point-of-care (POC) tests aimed at detecting celiac disease antibodies have been developed. It has been suggested that these POC tests can be implemented as a replacement for standard laboratory tests. OBJECTIVE: To evaluate the diagnostic accuracy of a POC test (Simtomax®) that detects celiac disease antibodies compared with standard laboratory tests when screening for celiac disease among patients referred for fertility treatment in 2 Danish fertility clinics. METHODS: Serum samples were analyzed for IgA anti-tissue transglutaminase (TGA) as the reference standard test with a cutoff of ≥7 kU/L and by the index POC test based on IgA and IgG antibodies against deamidated gliadin peptides (DGP). In IgA deficiency, the reference standard test was IgG DGP with a cutoff of ≥7 kU/L. Participants answered a questionnaire on gluten intake, symptoms, and risk factors. Diagnostic confirmation was made by duodenal biopsies. IgA TGA/IgG DGP were used as the reference standard to calculate positive and negative predictive values. RESULTS: A total of 622 men and women (51.6%) were enrolled during 2015. The reference standard IgA TGA/IgG DGP was positive in 7 participants (1.1% [95% CI 0.5-2.3]) and the POC test was positive in 84 participants (13.5% [95% CI 10.9-16.4]), 3 of whom also had positive reference standard tests. This yields a sensitivity of the index POC test of 42.9% (95% CI 9.9-81.6) and a specificity of 86.8% (95% CI 83.9-89.4). Positive and negative predictive values were 3.57% (95% CI 0.7-10.1) and 99.3% (95% CI 98.1-99.8). CONCLUSION: The sensitivity of the POC test was low; however, the specificity was moderately good. The POC test had a high negative predictive value in this low prevalent population but missed 1 patient with biopsy-confirmed celiac disease. However, because of many false-positive tests, it cannot be recommended as replacement for standard laboratory tests but rather as a triage test to decide if the standard serology tests should be performed.

Therapeutic endometrial scratching and implantation after in vitro fertilization: a multicenter randomized controlled trial.

OBJECTIVE: To study whether endometrial scratching in the luteal phase before ovarian stimulation increases clinical pregnancy rates in women with one or more previous implantation failures. DESIGN: A nonblinded multicenter randomized clinical trial. SETTING: Fertility clinics. PATIENT(S): Three hundred four eligible patients scheduled for IVF/intracytoplasmic sperm injection were randomized. The intervention group (n = 151) underwent endometrial scratching in the luteal phase before controlled ovarian stimulation, while no intervention was performed in the control group (n = 153). INTERVENTION(S): Endometrial scratching with a Pipelle de Cornier catheter in the luteal phase before ovarian stimulation. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate and prenatal and birth data. RESULT(S): There was no overall significant improvement in clinical pregnancy rates between the control and intervention groups (38.5% vs. 44.4%; relative risk = 1.15; confidence interval [0.86-1.55]). However, subgroup analyses revealed that women with three or more previous implantation failures had a significant increase in clinical pregnancy rate (31.1% vs. 53.6%; relative risk = 1.72; confidence interval [1.05-2.83]) after scratching. No difference was seen as regards prenatal and birth data between the two groups. CONCLUSION(S): Endometrial scratching in the luteal phase before ovarian stimulation significantly enhances the clinical pregnancy rate in women with three or more prior implantation failures. This result seems to corroborate previous reports, which found that particularly women with repeated implantation failure seem to gain a positive effect from endometrial scratching. Importantly, there were no significant differences in prenatal data and birth data between the groups. CLINICAL TRIAL REGISTRATION NUMBER: NCT01963819.

Unrecognised coeliac disease among men and women undergoing fertility treatment: A screening study.

BACKGROUND: Coeliac disease is an autoimmune disease triggered by dietary gluten and has been associated with several conditions influencing female and male reproduction. Due to unspecific symptoms, coeliac disease can be unrecognised for years. OBJECTIVE: To estimate the prevalence of unrecognised coeliac disease among couples referred to fertility treatment. METHODS: Cross-sectional screening for coeliac disease in men and women referred to fertility treatment using IgA tissue transglutaminase antibodies as a marker of coeliac disease and small-bowel biopsies to confirm the diagnosis. Participants answered a questionnaire on gluten intake, gastrointestinal symptoms and reproductive history. RESULTS: A total of 893 participants (51% women) were screened and eight were coeliac disease antibody positive. Small-bowel biopsies were obtained from seven antibody positive participants and unrecognised coeliac disease was confirmed in one woman and three men, corresponding to a prevalence of 0.45% (95% confidence interval 0.12-1.14). The total prevalence, combining already diagnosed and unrecognised CD cases, was 0.63% (95% confidence interval 0.29-1.12). CONCLUSION: The prevalence of unrecognised coeliac disease in a group of infertile patients was equivalent to that of the Danish general population and low compared with that observed in the majority of other screening studies of infertile patients. Surprisingly, it should be noted that more men than women had coeliac disease. This result does not support a need for routine screening among infertile patients.

Prevalence, incidence, and autoimmune comorbidities of celiac disease: a nation-wide, population-based study in Denmark from 1977 to 2016.

AIM: The aim of this study was to describe and identify potential trends with respect to prevalence, incidence, age, sex, and autoimmune comorbidity of celiac disease (CD). PATIENTS AND METHODS: A Danish nationwide cohort study of CD using data from The National Patient Register. Patients with a primary or secondary diagnosis code of CD during the period 1977 to 2016 were identified. Information on sex, date of birth, death, or immigration was obtained from the Danish Civil Registration System, and autoimmune comorbidities were identified in the Danish National Patient Register. The CD cohort was compared with the general Danish population using a control cohort and aggregated data obtained from Statistics Denmark. RESULTS: The CD cohort consisted of 11 802 (65% women) patients. The median age at diagnosis of CD varied between 30 years in 1980-1984 and 45 years in 1995-1999 and 27 years in 2015-2016. The prevalence of CD in 1986 and 2016 was 14 and 180 per 100 000 persons, respectively, with a female/male ratio changing from 1.3 to 2.0. Incidence rates (per 100 000 person-years) changed from 1.6 in 1980-1984 to 15.2 in 2015-2016, with the largest increase among females aged 0-9 years. In 2016, prevalence of autoimmune comorbidities was 16.4% among the CD patients compared with 5.3% in the general population. CONCLUSION: The prevalence of diagnosed CD has doubled every decade in Denmark from 1986 to 2016, and in the same period the female/male ratio has increased and the median age at diagnosis has decreased. The prevalence of autoimmune comorbidity in 2016 was three times higher among CD patients compared with the general Danish population.

Effect of women's age on embryo morphology, cleavage rate and competence-A multicenter cohort study.

This multicenter cohort study on embryo assessment and outcome data from 11,744 IVF/ICSI cycles with 104,830 oocytes and 42,074 embryos, presents the effect of women's age on oocyte, zygote, embryo morphology and cleavage parameters, as well as cycle outcome measures corrected for confounding factors as center, partner's age and referral diagnosis. Cycle outcome data confirmed the well-known effect of women's age. Oocyte nuclear maturation and proportion of 2 pro-nuclear (2PN) zygotes were not affected by age, while a significant increase in 3PN zygotes was observed in both IVF and ICSI (p<0.0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first time, we show that a woman's age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate, if this increase in initial hCG value with advancing maternal age is connected to the embryo or the uterus.

Microarray-Based Analysis of Methylation of 1st Trimester Trisomic Placentas from Down Syndrome, Edwards Syndrome and Patau Syndrome.

Methylation-based non-invasive prenatal testing of fetal aneuploidies is an alternative method that could possibly improve fetal aneuploidy diagnosis, especially for trisomy 13(T13) and trisomy 18(T18). Our aim was to study the methylation landscape in placenta DNA from trisomy 13, 18 and 21 pregnancies in an attempt to find trisomy-specific methylation differences better suited for non-invasive prenatal diagnosis. We have conducted high-resolution methylation specific bead chip microarray analyses assessing more than 450,000 CpGs analyzing placentas from 12 T21 pregnancies, 12 T18 pregnancies and 6 T13 pregnancies. We have compared the methylation landscape of the trisomic placentas to the methylation landscape from normal placental DNA and to maternal blood cell DNA. Comparing trisomic placentas to normal placentas we identified 217 and 219 differentially methylated CpGs for CVS T18 and CVS T13, respectively (delta ß>0.2, FDR<0.05), but only three differentially methylated CpGs for T21. However, the methylation differences was only modest (delta ß<0.4), making them less suitable as diagnostic markers. Gene ontology enrichment analysis revealed that the gene set connected to theT18 differentially methylated CpGs was highly enriched for GO terms related to"DNA binding" and "transcription factor binding" coupled to the RNA polymerase II transcription. In the gene set connected to the T13 differentially methylated CpGs we found no significant enrichments.

Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

The parental origin correlates with the karyotype of human embryos developing from tripronuclear zygotes.

OBJECTIVE: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. METHODS: The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. RESULTS: 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. CONCLUSION: The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization.

OBJECTIVE: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). DESIGN: Multicenter, randomized, placebo-controlled, double-blinded prospective design. SETTING: Fourteen Scandinavian fertility clinics. PATIENT(S): A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). INTERVENTION(S): Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. MAIN OUTCOME MEASURE(S): OIR at gestational week 7, with follow-up at week 12 and birth. RESULT(S): At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. CONCLUSION(S): Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. CLINICAL TRIAL REGISTRATION NUMBER: NCT00565747.

Limited implantation success of direct-cleaved human zygotes: a time-lapse study.

OBJECTIVE: To evaluate embryos with direct cleavage (≤5 hours) from two to three cells (DC2-3) and correlate this morphokinetic parameter to implantation and ongoing pregnancy. DESIGN: Clinical multicenter retrospective study. SETTING: Private in vitro fertilization (IVF) centers. PATIENT(S): From three clinics, a total of 979 treatments including 5,225 embryos using autologous or donated oocytes, of which 1,659 embryos were transferred. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy confirmed by ultrasound in week 7. RESULT(S): Of the total embryo cohort, 715 (13.7%) underwent direct cleavage from two to three cells, 1,659 embryos were transferred to recipients, and 109 of the transferred embryos cleaved directly from two to three cells (6.6%). Only one DC2-3 embryo was known to result in a clinical pregnancy (1%) and 80 (73.4%) DC2-3 embryos did not implant. Of the 1,550 embryos transferred not showing DC2-3, 203 embryos were from treatments with 100% implantation (13.1%), and 804 (51.8%) embryos did not implant. The known implantation rate of DC2-3 embryos was statistically significantly lower than for embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively). CONCLUSION(S): Embryos with DC2-3 had a statistically significantly lower implantation rate than embryos with a normal cleavage pattern, suggesting that rejection of these embryos for transfer could improve the implantation rate.