Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study.

BACKGROUND: This study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5). METHODS: From September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes. RESULTS: In single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05). CONCLUSION: When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

Association between blood lead levels and unfavorable IVF outcomes: potential involvement of endoplasmic reticulum stress response in granulosa cells.

PURPOSE: To investigate the relationship between blood lead levels (BLLs) and IVF clinical outcomes in infertile females and to further explore the possible involvement of granulosa cell (GC) endoplasmic reticulum (ER) stress in the process. METHODS: One hundred twenty-three infertile women undergoing IVF cycles were included in the current study. All participants were divided into three (low, medium, and high) groups determined by BLL tertiles. Gonadotropin releasing hormone (GnRH) agonist regimen for ovarian stimulation was used for all patients, with follicular fluids being collected on the day of oocyte retrieval. Lactate dehydrogenase (LDH) levels in follicular fluid and the endoplasmic reticulum stress-signaling pathway of granulosa cells (GCs) were examined. RESULTS: The oocyte maturation rate and high-quality embryo rate on cleaved stage decreased significantly as BLL increased. For lead levels from low to high, live birth rate (68.29%, 56.10%, 39.02%; P=0.028) showed negative correlations with BLLs. Also, follicular fluid Pb level and LDH level was significantly higher in the high lead group versus the low group. Binomial regression analysis revealed significant negative correlation between BLLs and live birth rate (adjusted OR, 0.38; 95% CI, 0.15-0.95, P=0.038). Further analysis of the endoplasmic reticulum stress (ER stress) signaling pathway of GCs found that expressions of GRP78, total JNK, phosphorylated JNK, and CHOP increased and BCL-2 decreased with increasing BLLs. CONCLUSIONS: BLLs are negatively associated with final clinical outcomes in IVF patients that may be related to increased ER stress response and GC apoptosis. Thus, reducing Pb exposure before IVF procedures may improve final success rates.

A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

Betanin inhibits PI3K/AKT/mTOR/S6 signaling pathway, cell growth and death in osteosarcoma MG-63 cells.

It is possible to develop new chemopreventive compounds so that cancer cells can be targeted in an exclusive manner. Bioactive natural compounds have demonstrated to be efficient chemotherapeutic agents, safe and cost-effective. Majority of anti-cancer medications are derived from natural sources, particularly of plant origins. Betanin (betanidin-5-O-ß-glucoside) is the most common betacyanin with antioxidant, anti inflammatory and anticancer properties. The present study therefore investigated the effect of betanin onosteosarcoma MG-63 cells. The mechanistic pathway of inflammatory responses, cell proliferation and apoptosis were investigated. The MG-63 cells were treated with betanin for 24 h. Betanin actions on the appearance of cell arrangements, morphological changes, ROS induced Δψm , cell migration, cell adhesion and proliferative mechanistic marker expression of PI3K/AKT/mTOR/S6were analyzed. Betanin inhibited MG-63 cells at IC50 concentrations between 9.08 and 54.49 µM and induced apoptosis by triggering the ROS mechanism. Betanin inhibited proliferation and migration of MG-63 cells and induced DNA fragmentation. Betanin also modified the key mediator expression levels of PI3K/AKT/mTOR/S6 signaling pathways. Betanin can potentially be utilized in bone carcinoma therapeutics to inhibit, reverse or delay osteosarcoma.

Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

Comparison of Pregnancy Outcomes Between Single-Morula Embryo Transfer and Single-Blastocyst Transfer in Fresh IVF/ICSI Cycles.

BACKGROUND This study investigated the effectiveness and feasibility of day 4 (D4) morula embryo transfer (ET) in comparison with day 5 (D5) blastocyst ET, with regards to their clinical data, laboratory test results, and pregnancy outcomes. MATERIAL AND METHODS This retrospective cohort study enrolled 1070 patients, including 178 cases in group D4 and 892 cases in group D5. The endpoint was live birth rate after fresh embryo transfer. Furthermore, the clinical outcomes of D4 embryos with different morphology were compared and assigned to 3 groups: in group 1 (n=66) the embryos were compacted but not expanded, in group 2 (n=102) the embryos were compacted and expanded (early blastocyst), and in group 3 (n=10) the embryos were not compacted. RESULTS Groups D4 and D5 had comparable clinical pregnancy rates (53.37% vs. 59.97%) and live birth rates (43.25% vs 50.89%), and there were no significant differences between the 2 groups. In group 3, there was only 1 clinical pregnancy and no live birth. In comparison between group 1 and group 2, the clinical pregnancy rate of group 2 showed an upward trend (48.48% vs 60.78%), but there was no significant difference. There was also no statistically significant difference in the live birth rate between the 2 groups (42.42% vs 49.01%). CONCLUSIONS Transferring of compacted embryos or early blastocysts can result in high clinical pregnancy rates and live birth rates. In addition to the cleavage and blastocyst ET, morula ET may serve as an alternative option for the clinician.

Prevalence of problematic Internet use and its association with quality of life among undergraduate nursing students in the later stage of COVID-19 pandemic era in China.

BACKGROUND AND OBJECTIVES: The prevalence of problematic Internet use (PIU) in the post-COVID-19 pandemic era is not known. This cross-sectional study aimed to determine the prevalence of PIU among baccalaureate nursing students (hereafter: nursing students) in the post-COVID-19 era. METHODS: A total of 1070 nursing students were consecutively invited to participate in this study from the nursing schools of five universities. PIU and quality of life (QOL) were assessed using the Internet Addiction Test (IAT) and the World Health Organization Quality of Life Scale Brief Version (WHOQOL-BREF), respectively. t Tests, χ2 , tests, and Kruskal-Wallis tests were used to compare basic demographic and clinical characteristics between participants with and without PIU. Binary logistic regression analysis was used to examine independent correlates. RESULTS: The prevalence of PIU was 23.3% (95% confidence interval [CI]: 20.7%-25.8%). Multiple logistic regression analysis revealed that second- (p = .024) and third-year (p = .012) students were more likely to suffer from PIU compared with first year students. Students with more severe depressive (p = .014) and anxiety symptoms (p = .011) were independently and significantly associated with more severe PIU. After controlling for covariates, nursing students with PIU had a lower overall QOL score (p = .002). CONCLUSION AND SCIENTIFIC SIGNIFICANCE: Problematic Internet use (PIU) was common among nursing students in the post-COVID-19 era. Considering the negative impact of PIU on QOL and academic performance, regular screening should be conducted and effective interventions implemented for nursing students with PIU. This was the first study on the prevalence of PIU among nursing students in the post-COVID-19 era. The findings of this study could help health professionals and education authorities to understand the patterns of PIU and its influence on QOL among nursing students and to allocate health resources and develop effective measures to reduce the risk of PIU in this population.

Assisted oocyte activation with calcium ionophore 44 hours after intracytoplasmic sperm injection resulting in successful pregnancy.

Assisted Oocyte Activation (AOA) with Calcium Ionophore, is possible to manually activate the oocytes and cure globozoospermia, thus leading to successful pregnancy in 1 h after ICSI. But in this case, we report a case that 44 h after ICSI, the arrest zygotes assisted oocyte activation with calcium ionophore, obtained clinical pregnancy and live birth. Accordingly, AOA may provide us with an immediate treatment for embryonic arrest in the future.

A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.

Energy requirements in mammalian oogenesis.

Oogenesis is a lengthy, multi-step process occurring in mammals yielding single or multiple oocytes capable of being fertilized upon interaction with male gametes. The overall process is highly complex in nature, starting in the primordial follicles, and its ultimate completion is preceded by the meiotic cycle. There are two major phases in oogenesis: the growth phase, followed by a maturation phase that requires relatively less time. Both phases require energy for the various metabolic processes of the oocytes. The energy requirements and the timing of maturation vary significantly among mammalian species. This review describes the variations in the mammalian oocytes development and their energy requirements. It covers the types of mitochondria, the distribution of their changes, and the metabolic processes occurring during the oogenesis in different mammalian species. Oocyte abnormalities associated with glucose deficiency in mammals are discussed, along with the role of fat and protein as alternative energy substrates. The review concludes with recommendations for future studies on oogenesis in mammalian species in the context of energy requirements.

The effect of protein supplement concentration in embryo transfer medium on clinical outcome of IVF/ICSI cycles: a prospective, randomized clinical trial.

The aim of this prospective, randomized clinical trial (RCT) was to evaluate whether the supplemental protein concentration in embryo transfer (ET) medium affects the clinical outcomes in IVF-ET. A total of 750 patients undergoing IVF-ET who met the inclusion criteria were randomly divided into three groups, according to the concentration of synthetic serum substitute (SSS) in ET medium as follows: 10% (Group A), 20% (Group B) and 50% (Group C). The patient characteristics and embryology data were all similar among the groups. The rates of implantation, clinical pregnancy and live birth were compared. Clinical pregnancy (44.61%, 48.79% and 45.49%), multiple pregnancy (24.18%, 28.71% and 25.0%), implantation (28.21%, 30.68% and 29.86%) and live birth (41.67%, 43.96% and 41.70%) rates in the three groups (A, B and C, respectively) showed no significant differences. This RCT demonstrates that supplemental protein concentration in the ET medium does not affect the treatment outcomes in IVF-ET. There was no statistical evidence to support the hypothesis that supplemental protein concentration in the ET medium influences treatment outcomes in IVF-ET.

Effect of testosterone deficiency on cholesterol metabolism in pigs fed a high-fat and high-cholesterol diet.

BACKGROUND: Testosterone deficiency is associated with increased serum cholesterol levels. However, how testosterone deficiency precisely affects cholesterol metabolism remains unclear. Therefore, in the current study, we examined the effect of testosterone deficiency on cholesterol metabolism and liver gene expression in pigs fed a high-fat and high-cholesterol (HFC) diet. METHODS: Sexually mature male miniature pigs (6-7 months old) were randomly divided into 3 groups as follows: intact male pigs fed an HFC diet (IM+HFC), castrated male pigs fed an HFC diet (CM+HFC), and castrated pigs with testosterone replacement fed an HFC diet (CM+HFC+T). Serum testosterone levels and lipid profiles were measured, and gene expression levels associated with hepatic cholesterol metabolism were determined. Furthermore, total hepatic cholesterol contents and the activities of enzymes mediating hepatic cholesterol metabolism were measured. RESULTS: Serum testosterone levels were significantly decreased in CM+HFC pigs, and testosterone replacement attenuated castration-induced testosterone deficiency. Castration significantly increased the serum levels of total cholesterol, low-density lipoprotein cholesterol and triglycerides, as well as hepatic lipid contents in pigs fed an HFC diet. Compared with IM+HFC and CM+HFC+T pigs, low-density lipoprotein receptor (LDLR) mRNA expression and protein levels were significantly decreased in the livers of CM+HFC pigs. In contrast, we found that compared with IM+HFC pigs, hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and serum PCSK9 protein levels were significantly increased in CM+HFC pigs. Moreover, testosterone treatment reversed the increase in PCSK9 expression in CM+HFC pigs. However, neither castration nor testosterone replacement affected the expression of the other hepatic genes that were tested. CONCLUSIONS: This study demonstrated that castration-induced testosterone deficiency caused severe hypercholesterolemia in pigs fed an HFC diet; furthermore, these effects could be reversed by testosterone replacement therapy. Altered hepatic PCSK9 and LDLR expression, resulting in reduced LDL-cholesterol clearance, may contribute to the increased serum cholesterol levels induced by testosterone deficiency and an HFC diet. These results deepen our understanding of the underlying molecular mechanisms that mediate the effects of testosterone deficiency on cholesterol metabolism.

Blastocyst-stage versus cleavage-stage embryo transfer in the first frozen cycles of OHSS-risk patients who deferred from fresh embryo transfer.

Elective cryopreservation of all embryos has been the most effective means to avoid developing ovarian hyperstimulation syndrome (OHSS). However, it is still unknown which stage is optimal for freezing and transferring into uterus in OHSS-risk patients. This study was undertaken to evaluate whether OHSS-risk patients could benefit from transferring blastocysts. A total of 162 women were allocated to cleavage-stage embryo transfer (ET) (group A = 70) and blastocysts transfer (group B = 92) on the basis of patients' voluntary in their first frozen cycles. Although the mean number of transferred embryos in group A was significantly more than those in group B (2.37 ± 0.52 versus 2.11 ± 0.52, p < 0.05), the clinical pregnancy rates, implantation rates and live birth rates in group B were significantly higher than those in group A (47.83% versus 31.43%, p < 0.05; 31.44% versus 18.67%, p < 0.05; 40.21% versus 27.14%, p < 0.05), and the multiple pregnancy rates in both groups were comparable (34.09% versus 36.36%, p > 0.05). The observed results in OHSS-risk population allow us to take a position in favor of blastocyst transfer, thus pregnancy and live birth could be achieved with fewer ETs and in a shorter time frame.

Live birth following vitrification of in vitro matured oocytes derived from sibling smaller follicles at follicle selection phase in the context of in vitro fertilization.

In ovarian stimulation, a 31-year-old woman with polycystic ovary syndrome was at the risk of developing ovarian hyperstimulation syndrome, follicle aspiration was performed, and eight immature oocytes were collected from follicle fluids. After 28 h in vitro culture, six of them reached MII and were vitrified. The patient failed to conceive in her fresh in vitro fertilization cycle and next two replacement cycles. In the third replacement cycle, a successful pregnancy was obtained by vitrified-thawed oocytes. This case demonstrates that follicular aspiration during follicle selection phase has protective effects against developing ovarian hyperstimulation syndrome, and rescued immature oocytes are viable and could produce promising embryos for live birth.

Chronic hepatitis B virus infection in women is not associated with IVF/ICSI outcomes.

OBJECTIVE: This study was to evaluate whether chronic HBV (Hepatitis B virus) infection in women is associated with poor performance following IVF/ICSI (in vitro fertilization/intracytoplasmic sperm injection) treatments. MATERIALS AND METHODS: 123 cycles with female chronic HBV infection were compared with 246 cycles with no-infected couples, matched for female age, D3 serum FSH (follicles stimulation hormone) levels, body mass index and assisted reproductive technology approach used (IVF or ICSI), in a ratio of 1:2. RESULTS: The details in IVF/ICSI cycles, including the dosage of gonadotrophin used, the serum estradiol levels and the endometrial thickness on the day of hCG (human chorionic gonadotrophin) injection, the mean number of oocytes retrieved, and the embryology data, were similar among seropositive and seronegative women. And there was no significant differences in implantation rates and live birth rates between seropositive women group and matched control (30.52 versus 28.34% per transfer; 42.28 versus 40.65%). CONCLUSIONS: The results indicated that women with chronic HBV infection is not associated with outcomes of IVF/ICSI treatments.

P407 hydrogel loaded with nitric oxide microbubbles promotes angiogenesis and functional improvement in testicular transplantation.

Prepubertal male patients with cancer have decreased fertility after treatment, but there are currently no suitable means for fertility rescue. Testicular transplantation seems to be a promising treatment. The short-term insufficiency of blood supply after transplantation is the key problem that needs to be solved. In this research, nitric oxide (NO), a gas and small molecule transmitter with the effect of promoting angiogenesis, acted at the site of testicular transplantation. Herein, poloxamer-407 (P407) and lipid microbubble materials served as transport carriers for NO and helped NO to function at the transplant site. P407 hydrogel loaded with NO microbubbles (PNO) slowly released NO in vitro. The three-dimensional space of the hydrogel provided a stable environment for NO microbubbles, which is conducive to the continuous release of NO. In this study, 25% PNO (w/v) was selected, and the gelling temperature was 19.47 °C. The gelling efficiency was relatively high at body temperature. Rheological experiments showed that PNO, at this concentration, had stable mechanical properties. The results from in vivo experiments demonstrated that testicular grafts in the PNO group exhibited a notably accelerated blood flow recovery compared to the other groups. Additionally, the PNO group displayed a significant improvement in reproductive function recovery. In conclusion, PNO exhibited slow release of NO, and a small amount of NO promoted angiogenesis in testicular grafts and restored reproductive function.

Enhancing oocyte in vitro maturation and quality by melatonin/bilirubin cationic nanoparticles: A promising strategy for assisted reproduction techniques.

In assisted reproduction techniques, oocytes encounter elevated levels of reactive oxygen species (ROS) during in vitro maturation (IVM). Oxidative stress adversely affects oocyte quality, hampering their maturation, growth, and subsequent development. Thus, mitigating excessive ROS to safeguard less viable oocytes during IVM stands as a viable strategy. Numerous antioxidants have been explored for oocyte IVM, yielding considerable effects; however, several aspects, including solubility, stability, and safety, demand attention and resolution. In this study, we developed nanoparticles by self-assembling endogenous bilirubin and melatonin hormone coated with bilirubin-conjugated glycol chitosan (MB@GBn) to alleviate oxidative stress and enhance oocyte maturation. The optimized MB@GBn exhibited a uniform spherical shape, measuring 128 nm in particle size, with a PDI value of 0.1807 and a surface potential of +11.35 mV. The positively charged potential facilitated nanoparticle adherence to the oocyte surface through electrostatic interaction, allowing for functional action. In vitro studies demonstrated that MB@GB significantly enhanced the maturation of compromised oocytes. Further investigation revealed MB@GB's effectiveness in scavenging ROS, reducing intracellular calcium levels, and suppressing mitochondrial polarization. This study not only offers a novel perspective on nano drug delivery systems for biomedical applications but also presents an innovative strategy for enhancing oocyte IVM.

DMAP-catalyzed [3 + 2] and [4 + 2] cycloaddition reactions between [60]fullerene and unmodified Morita-Baylis-Hillman adducts in the presence of Ac2O.

One-step DMAP-catalyzed [3 + 2] and [4 + 2] cycloaddition reactions between C(60) and unmodified Morita-Baylis-Hillman adducts in the presence of Ac(2)O have been developed for the easy preparation of cyclopentene- and cyclohexene-fused [60]fullerene derivatives. When the MBH adducts bear an alkyl group, two different reaction pathways could be controlled selectively depending on the conditions.

[Suppression of mitochondrial oxidative phosphorylation on in vitro maturation, fertilization and developmental competence of oocytes].

OBJECTIVE: To investigate the roles of mitochondrial oxidative phosphorylation (OXPHOS) capacity in oocyte maturation, fertilization and embryo development. METHODS: Carbonyl cyanide p- (tri-fluromethoxy) phenyl-hydrazone (FCCP), a metabolic inhibitor of mitochondria, was introduced into culture medium. The integrity of spindle and chromosome alignment, reactive oxygen species (ROS) levels, and rates of maturation, germinal vesicle breakdown, fertilization and blastulation were assessed in vitro. RESULTS: Significant decreases were detected in the percentages of oocytes with nuclear maturation, normal spindle formation and chromosome alignment, ROS levels and capable for blastocyst formation between oocytes treated with FCCP and non-treated (control group), 55.8%, 37.9%, 0.67 and 57.9% (FCCP 10 nmol/L group), 47.3%,34.7%, 0.59 and 41.8% (FCCP 100 nmol/L group) versus 62.9%, 61.9%,0.94 and 68.3% (control group) respectively, P<0.05. However, No significant differences were found in the rates of GVBD and fertilization in oocytes from the FCCP treated and the control. CONCLUSION: Inhibition of mitochondrial metabolic capacity resulted in decreased the percentages of oocytes with nuclear maturation, normal spindle formation and chromosome alignment, ROS levels and capable for blastocyst formation. But the treatment of FCCP did not affect the rate of fertilization.

[Effects of controlled ovarian hyperstimulation on mitochondria copy number and functions in murine oocytes].

OBJECTIVE: To investigate the effect of whether controlled ovarian hyperstimulation (COH) on mitochondrial DNA (mtDNA) copy number, mitochondrial function, distribution, the level of reactive oxygen species (ROS) in oocytes and the mechanism of oocyte loss in COH. METHODS: Matured murine oocytes were classified into COH group and natural cycles (NC) group. The copies of mtDNA, the magnitude of mitochondrial membrane potential (Δφm) and oocyte adenosine triphosphate (ATP) content, pattern of mitochondrial distribution, and ROS levels were evaluated by realtime PCR, immunofluorescence and fluorescence-luciferase mensuration. RESULTS: The copies of mtDNA, the levels of Δφm, and ATP content in oocytes between COH and NC groups showed statistical difference [(1.15 ± 0.01)×10(5), 0.34 ± 0.03 and (241 ± 20) fmol/oocyte (COH)] versus [(2.15 ± 0.19)×10(5), 0.82 ± 0.07 and (325 ± 11) fmol/oocyte (NC)], respectively(P < 0.05). However, there were no significant differences in the rate of evenly distributed mitochondria and the level of ROS in oocytes from COH and NC [(76.5% (78/102) in COH versus 82.1% (69/84)]; 1.07 ± 0.07 in COH versus 0.93 ± 0.08 in NC (P > 0.05). CONCLUSION: It was indicated that non-physiological COH treatments inhibit mtDNA replication, alter mitochondrial function, which might partly be involved in the low development potential of COH oocyte.