Oral administration of l-carnitine improves the clinical outcome of fertility in patients with IVF treatment

Age-dependent decline of mitochondrial function has been proposed to be a main cause of decline of embryo quality. Then, l-carnitine plays important roles in reducing the membranous toxicity of free-fatty acids by forming acyl-carnitine and promoting β-oxidation, preventing cell damage. Recent research revealed that l-carnitine played important roles in vitro in oocyte growth, oocyte maturation and embryo development. However, such beneficial effects of l-carnitine in vivo have yet to be verified. The effect of oral l-carnitine supplementation on embryo quality and implantation potential was examined. A total of 214 patients were included in this study. They all previously received in vitro fertilization-embryo transfer (IVF-ET) and failed to conceive. Then they were administered l-carnitine for 82 days on average and underwent IVF-ET again. There were no significant differences in the total number of retrieved oocytes, and their maturation and fertilization rates between before and after l-carnitine administration. The quality of embryos on Days 3 and 5 after insemination was improved following l-carnitine administration (p?l-carnitine administration compared with previous cycles. Healthy neonates were born after IVF-ET following l-carnitine administration. Our data suggested that oral administration of l-carnitine to fertility patients improved the developmental competence of their oocytes after insemination.     

Lowered serum total L-carnitine levels are associated with obesity at term pregnancy

Abstract

Objective: This study aims to compare the serum total l-carnitine concentrations of obese and non-obese pregnant women and to identify the role of l-carnitine in both maternal and fetal weight gain during pregnancy.

Method: This study reviews 118 healthy women with singleton term pregnancy (≥37 weeks). The characteristics of the recruited subjects were analyzed according to their pre-pregnancy body mass index (BMI).

Results: The women with pre-pregnancy BMI?<?18.5?kg/m² had significantly higher serum l-carnitine levels whereas the women with BMI?>?29.9?kg/m² at term pregnancy had significantly lower serum l-carnitine levels (p?=?0.001 for both). The neonates born to women with BMI?>?29.9?kg/m² at term pregnancy had significantly longer height and wider head circumference (p?=?0.001 for both). Serum total l-carnitine levels correlated significantly and negatively with pre-pregnancy body weight, pre-pregnancy BMI, pregnancy body weight, pregnancy BMI and serum triglyceride levels (r?=??0.397, p?=?0.001; r?=??0.357, p?=?0.001; r?=??0.460, p?=?0.001; r?=??0.463, p?=?0.001 and r?=??0.216, p?=?0.019, respectively). There was a significant and positive correlation between l-carnitine and HDL values (r?=?0.243, p?=?0.008).

Conclusions: The crucial role of l-carnitine in pregnancy metabolism suggests that nutritional supplementation of this amino acid can be offered to women who are either overweight or obese at the beginning of the pregnancy.     

Quantitative and qualitative changes of mitochondria in human preimplantation embryos

Purpose

The oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure.

Methods

Sixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2–14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos.

Results

The structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells.

Conclusions

The mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

     

<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine: a new insight into the pathogenesis of endometrial cancer

Objectives

The present study aims to specify the role of l-carnitine in the pathogenesis of endometrial cancer by comparing the serum total l-carnitine levels of endometrial cancer patients with those of healthy women.

Methods

Serum total l-carnitine concentrations were measured in patients with endometrioid-type endometrial cancer (n = 20) and healthy controls (n = 20) who were matched with respect to age and body mass index (BMI).

Results

Stage I endometrial cancer was diagnosed in 12 women (60.0 %) whereas three women (15.0 %) had stage II disease, three women (15.0 %) had stage III disease and two women (10.0 %) had stage IV disease. The healthy controls and endometrial cancer patients were statistically similar in aspect of age, gravidity, parity, BMI, waist-to-thigh ratio, waist-to-hip ratio, menopause, complete blood count parameters, and serum biochemistry. Serum total l-carnitine levels of women with endometrial cancer were significantly lower than those of healthy women (respectively, 5,519.4 ± 2,712.5 vs 7,940.8 ± 3,566.6 ng/dl, p = 0.021). Moreover, serum total l-carnitine levels decreased significantly and progressively with advancing stage (stage I vs II vs III vs IV; 6,294.0 ± 2,885.1 vs 5,800.0 ± 441.2 vs 4,016.0 ± 2,833.3 vs 2,560.0 ± 67.9 ng/dl; p = 0.021).

Conclusions

This is the first study to hypothesize that l-carnitine deficiency participates in the pathogenesis of endometrial cancer by means of a mechanism which is unrelated with obesity and increased amount of fat in human body.

     

Vaginal versus intramuscular progesterone for luteal phase support in assisted reproductive techniques: a systematic review and meta-analysis of randomized controlled trials

Abstract

We aimed to compare the efficacy of vaginal progesterone versus intramuscular progesterone (IMP) for luteal phase support in assisted reproductive techniques (ART). A comprehensive electronic search of four electronic databases (PubMed, Cochrane Library, Scopus, and ISI Web of Science) was performed from inception till August 2019 for randomized controlled trials (RCTs). We included studies performed different ART with the use of vaginal progesterone versus IMP for luteal phase support. Our primary outcome was clinical pregnancy rate. Our secondary outcomes were ongoing pregnancy, miscarriage, live birth rates, and satisfaction in both groups. 15 RCTs met our inclusion criteria with a total of 5656 patients. Our analysis indicated no significant differences between vaginal progesterone and IMP regarding clinical and ongoing pregnancies (RR = 0.90, 95% CI [0.80, 1.00], p?=?.06), (RR = 0.90, 95% CI [0.76, 1.06], p?=?.21), respectively. No significant differences were found between both routes of progesterone in miscarriage (p?=?.98) and live birth (p?=?.99). Subgroup analysis between fresh and frozen embryo transfer cycles in above outcomes showed no difference between progesterone routes. Vaginal progesterone was significantly associated with more satisfaction compared to IMP (p?<?.00001). In conclusion, vaginal progesterone can be used an alternative method for luteal phase support instead of IMP in ART.     

Modulatory effects of l-carnitine plus l-acetyl-carnitine on neuroendocrine control of hypothalamic functions in functional hypothalamic amenorrhea (FHA)

Functional hypothalamic amenorrhea (FHA) is a relatively frequent disease due to the combination of metabolic, physical, or psychological stressors. It is characterized by the low endogenous GnRH-induced gonadotropin secretion, thus triggering the ovarian blockade and a hypoestrogenic condition. Up to now various therapeutical strategies have been proposed, both using hormonal treatment as well as neuroactive compounds. Since carnitine, namely l-acetyl-carnitine (LAC), has been demonstrated to be effective in the modulation of the central hypothalamic control of GnRH secretion, we aimed to evaluate whether a combined integrative treatment for 12?weeks of LAC (250?mg/die) and l-carnitine (500?mg/die) was effective in improving the endocrine and metabolic pathways in a group of patients (n?=?27) with FHA. After the treatment, interval mean LH plasma levels increased while those of cortisol and amylase decreased significantly. When patients were subdivided according to baseline LH levels, only hypo-LH patients showed the significant increase of LH plasma levels and the significant decrease of both cortisol and amylase plasma levels. The increased 17OHP/cortisol ratio, as index of the adrenal activity, demonstrated the reduced stress-induced adrenal activity. In conclusion, our data sustain the hypothesis that the integrative administration of LAC plus l-carnitine reduced both the metabolic and the neuroendocrine impairment of patients with FHA.     

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching

Purpose

To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development.

Methods

Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy.

Results

PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos’ outer surface.

Conclusions

PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.     

Adrenomedullin Requires an Intact Nitric Oxide System to Function as an Endogenous Vasodilator in Rat Gestation

Objective: We hypothesize that administration of adrenomedullin (AM), an endogenous vasodilator, will ameliorate the hypertension and growth restriction associated with chronic nitric oxide inhibition induced by l‐ω nitro‐l‐arginine methyl ester (l‐NAME) infusion in pregnant rats. Methods. Osmotic minipumps were inserted on day 14 of gestation to deliver continuously either AM, l‐NAME, AM+l‐NAME, or vehicle control. Systolic blood pressure was recorded daily in pregnant rats. Pregnant rats were either sacrificed on gestational days 15, 16, 17, 18, or 22, or they were allowed to deliver at term. The placentas from all of the treated groups were fixed for 24 hr in Bouin solution, sectioned, processed, embedded in paraffin, and stained with hematoxylin and eosin. The placentas were graded for the quality of fetal vessel development in the labyrinth. Results. Systolic blood pressure was increased in AM+l‐NAME‐treated rats. The animals that delivered in the AM+l‐NAME group exhibited decreased pup weight (l‐NAME and AM+l‐NAME, 5.2±0.1 compared with 6.4±0.1 g for both AM and controls, p<0.001) and increased pup mortality (AM+l‐NAME, 44.4% compared with 16.7% in l‐NAME, 0% in AM and 3.1% in controls, p<0.001 AM+l‐NAME compared with controls). Increased decidual necrosis, necrosis in the labyrinth, and deficient fetal vessel development in the labyrinth was identified in the placentas treated with AM+l‐NAME. Conclusions. Addition of the endogenous vasodilator AM to an l‐NAME‐induced state of chronic NO inhibition did not ameliorate hypertension and growth restriction.     

Effect of parenterally l-arginine supplementation on the respiratory distress syndrome in preterm newborns

l-Arginine (l-Arg) is the precursor of nitric oxide which plays an important role on pulmonary circulation and pulmonary vascular tone. Earlier studies suggested that l-Arg levels in preterm newborns with respiratory distress syndrome (RDS) were low due to its consumption and l-Arg supplementation may reduce the severity of RDS. Our aim was detect the effect of the parenterally l-Arg supplementation on RDS severity. The subjects were chosen between preterm newborns (gestational age <34 weeks) (n?=?30). Twenty of the subjects were diagnosed with permaturity and RDS, and 10 of the subjects were healthy preterm newborns. Ten of the subjects was taken l-Arg (1.5?mmol/kg/d) in addition to routine RDS treatment and assumed as “Group 1”. In this group, daily l-Arg supplementation was started end of the first day, and continued at end of fifth day. The others of the subjects diagnosed with RDS was take routine RDS treatment and assumed as “Group 2”. Healthy preterm newbors assumed as “Group 3”. Blood collections for l-Arg levels via tandem mass spectrometry were made in first day and repeated on the seventh days. Oxygenation index was used to determine severity of RDS. l-Arg consentrations in Group 1 were 8.7?±?4.1?μM/L and 11.9?±?5.0?μM/L in first and seventh day, respectively. l-Arg consentrations were 12.6±4.5?μM/Land 10.9?±?5.4?μM/L in Group 2 and 8.6?±?5.1?μM/L and 9.4?±?4.1?μM/L in Group 3. There is no correlation between l-Arg concentrations and OI also duration of the mechanical ventilation of the subjects in patient groups (Group 1 and 2).     

Effect of l-arginine on blood pressure in pregnancy-induced hypertension: A randomized placebo-controlled trial

Objective.?To evaluate the antihypertensive efficacy of l-arginine (l-Arg) repeated infusions in women affected by gestational hypertension.

Methods.?The women were referred to obstetric units in order to assess their clinical conditions and to exclude the presence of severe fetal and/or maternal complications. Inclusion criteria were: maternal age range 16–45 years, diagnosis of gestational hypertension without proteinuria (patients normotensive until the 20th week), and gestational age ranging between 24 and 36 weeks. Each woman was allocated to receive either l-arginine (20 g/500 mL) or placebo treatment through an i.v. line. The infusion was carried out in the morning from 8 a.m. to 10 a.m. and it was repeated for the next four consecutive days. Systolic and diastolic blood pressure values as well as heart rate were recorded with the patient in an upright, seated position at 08:00, 12:00, 16:00 and 20:00 h.

Results.?Maternal clinical features such as age, height, weight, and gestational age at inclusion were similar between groups. Both systolic and diastolic blood pressures were reduced by treatment, the effect of l-arginine being significantly higher than that of the placebo (systolic values F = 8.59, p < 0.005; diastolic values F = 3.36; p < 0.001). Twenty women assigned to the l-Arg group (32.2%) and 23 to the placebo group (37.7%) were concomitantly treated with antihypertensives before starting the study. Analyzing the subgroup of patients not receiving antihypertensive drugs we found that l-arginine was superior to placebo in lowering systolic (F = 5.42, p < 0.005) and diastolic (F = 2.20, p < 0.005) blood pressure values.

Conclusions.?In conclusion, these data support the use of l-Arg as an antihypertensive agent for gestational hypertension especially in view of the other beneficial effects nitric oxide donors display in pregnancy. Further, l-Arg seems well tolerated since in this sample none of the patients reported adverse effects requiring study interruption.     

Effect of l-Arginine Supplementation on Blood Pressure in Pregnant Women: A Meta-Analysis of Placebo-Controlled Trials

Objective. A meta-analysis of placebo-controlled trials was conducted to evaluate the effect of l-arginine supplementation on blood pressure (BP) in pregnancy. Methods. Trials were searched in PubMed, Embase, and Cochrane Library. A total of five trials were included in the meta-analysis. Results. l-Arginine supplementation exhibited a mean decrease of 3.07 mmHg (p = 0.004) for diastolic blood pressure and a mean increase of 1.23 weeks (p = 0.002) for gestation age at delivery in pregnancy, but did not reduce systolic BP (p = 0.19) as compared to placebo. Conclusion. l-Arginine supplementation had a significant effect of lowering diastolic blood pressure and prolonging gestation age in pregnancy.     

Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated. Methods: Cumulus-oocyte-complexes (COCs) were matured in IVM medium with serum. Presumptive zygotes were cultured in serum free in vitro culture (IVC) medium. Embryos were used as chilled or control samples at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Embryos in 0.2 mL PBS in plastic straws were cooled rapidly in ethanol baths at 0°C for 30 min. Embryo viability was assessed by in vitro development. Results: The percentage of control embryos that hatched as blastocysts increased the later stage at which they were selected. Relative proportion of embryos increased from 28% to 48% to 68% when chilled at the 8-cell, morula or blastocyst stages. Conclusions: IVF-produced embryos are differentially susceptible to cooling injury. Cell counts made of those blastocysts formed from chilled embryos indicated subtle effects of chilling.     

Effect of L-arginine on the expression of Bcl-2 and Bax in the placenta of fetal growth restriction

Objective.?To investigate the effect of l-arginine on fetal growth restriction (FGR) in terms of the expression of Bcl-2 and Bax in placenta.

Methods.?Sixty pregnant women with FGR were randomized to receive conventional treatment alone (control group, n?=?30) or in combination with l-arginine (l-arginine group, n?=?30). The parameters of fetal growth and development were monitored by B-ultrasound at regular intervals. The newborn birth weight and perinatal outcomes were also documented. Placental tissue was sampled within 10?min after delivery for analysis. The expression of Bcl-2 and Bax in placental tissue was determined by immunohistochemical technique.

Results.?The fetal growth parameters of biparietal diameter, femur length, and abdominal circumference increased more significantly in l-arginine group than those in control group (p?<?0.0l). The cure rate and birth weight in l-arginine group were higher than those in control group (73.3% vs. 43.3%, 2455.20?g vs. 2402.63?g, respectively). The incidence of small for gestational age newborns in l-arginine group was significantly lower than that in control group. Compared with l-arginine group, the Bax expression increased, but bcl-2 expression decreased in control group.

Conclusions.?l-arginine could reduce the expression of Bax, and enhance the expression of bcl-2, which may be associated with reduced placental apoptosis and improved placental function and fetal development.     

Changes of red blood cell rheology in newborns with congenital hypothyroidism during treatment

Abstract

Aim: We aimed to evaluate the deformability characteristics of RBC and the affecting factors in newborns diagnosed with congenital hypothyroidism (CH) and to compare the outcomes after the l-thyroxin treatment.

Patients and methods: Enrolled subjects were divided into two subgroups as “patients” and age-matched healthy “controls”. First blood samples were taken from all subjects for measuring elongation index (rEI) and osmotic fragility of RBC (OF), hematic and biochemical analytes affecting the RBC deformability in the neonatal age. All parameters were repeated a month after provided euthyroid state following the treatment in patients and age-matched healthy controls.

Results: There was no difference between both groups in terms of complete blood count parameters and serum analytes (albumin, bilirubin and fibrinogen) except expected age-related changes in the first and second readings. Serum lipid/lipoprotein levels of both groups remained unchanged except triglyceride levels during the study period. The rEI of the patients were lower than that of controls in the first and second readings. The rEIs of the patients became increased, reaching (not equal) the levels of their controls during l-thyroxin treatment. Osmotic fragility of the patients was detected as lower than controls in the first and second readings, and became better during l-thyroxin treatment.

Conclusion: Our results indicate that some changes may occur on the hematic and biochemical analytes affecting the RBC deformability features. Neonates with CH have the worst rEI initially, but they reached the indices of the healthy infants thanks to l-thyroxin treatment. Also, their OF features have been improved by l-thyroxin.     

Treatment with d-chiro-inositol and alpha lipoic acid in the management of polycystic ovary syndrome

Abstract

To evaluate the effects of the combination of d-chiro inositol and alpha lipoic acid on menstrual cycles and insulin sensitivity in women with polycystic ovary syndrome (PCOS). Forty-one women with PCOS and 31 controls have been enrolled in the study. The menstrual cycle, BMI, homeostasis model assessment index (HOMA-I), and insulin secretion in response to an OGTT were evaluated before and after 6 months of treatment. During the observation period, the patients have been asked to not modify their diet and physical activity. The menstrual cycle length improved in 76.7% of the women. Ovulation was restored in 40%. During treatment, BMI significantly decreased (p<.002). The HOMA-I and insulin secretion were unchanged by treatment. However, when women were divided according to the presence of insulin resistance (IR; HOMA-I?>?2.5), in those with IR the HOMA-I and the insulin secretion significantly decreased (p<.05 and p<.006). The association of d-chiro-inositol and alpha lipoic acid improves menstrual cycle length, restoring ovulation in the majority of women. Insulin sensitivity improved in women with IR only, confirming that in presence of IR the d-chiro-inositol has a role in restoring the insulin action overcoming the inactivity of epimerase in transforming myo-inositol to d-chiro inositol.     

Asymmetric dimethylarginine and l-arginine levels in neonatal sepsis and septic shock

Objective: Nitric oxide (NO) formed by the enzyme NO synthase (NOS) from l-arginine, is an important mediator for pathogen elimination. Being a potent vasodilator NO is implicated in hypotension and decreased organ perfusion in sepsis. Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor. We investigated ADMA and l-arginine levels in neonatal sepsis and their relation to disease severity.

Methods: A prospective controlled study was conducted including 31 neonates with sepsis and 20 controls. Serum ADMA and l-arginine levels were measured within 24?h of sepsis diagnosis. Clinical and laboratory data including clinical risk index for babies (CRIB) score, presence of septic shock, organ dysfunction and death were recorded.

Results: l-Arginine and ADMA levels were higher in neonates with sepsis compared to controls (p?=?0.029 and p?=?0.001, respectively). Neonates with septic shock had higher ADMA levels compared to septic neonates without shock (p?=?0.026) and controls (p?l-Arginine levels were higher in neonates with septic shock compared to septic neonates without shock (p?=?0.012) and controls (p?l-arginine and ADMA levels. ADMA levels were correlated with CRIB score (rho?=?0.320, p?=?0.025).

Conclusion: l-arginine and ADMA levels are elevated in neonatal sepsis and even higher levels are observed in septic shock.     

The effect of cryopreservation on functional correlates of embryo integrity

To assess possible functional effects of the procedures involved in freezing/thawing of mammalian embryos, uptake of³H-glycine and¹²⁵I-BSA (bovine serum albumin) was measured in mouse two-cell embryos and morulae before and after freezing. None of the procedures of dehydration, rehydration, or freezing affected glycine uptake significantly. Earlier results for uptake of¹²⁵I-BSA by fresh embryos which indicated an increase in endocytic activity between morula and blastocyst stages were confirmed, and hydrolysis of endocytosed¹²⁵I-BSA by morulae but not two-cell embryos was observed. Only 53 ±9% of two-cell embryos but 83±7% of morulae were apparently normal after freezing and thawing. Apparently normal frozen/thawed two-cell embryos were twice as active in¹²⁵I-BSA uptake as controls. This was not the case for morulae. The results suggest that freezing/ thawing effects endocytotic processes in two-cell embryos but not morulae.     

Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.