Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer.

Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases.

The press-assisted fusion scheme significantly mitigates the quantity of HVJ-E required in mitochondrial replacement techniques.

In brief: The impact of HVJ-E employed in mitochondrial replacement techniques (MRTs) on embryonic development remains uncertain. This study has exhibited the influence of HVJ-E utilized in MRTs on embryonic development and has devised a novel HVJ-E-induced fusion approach to curtail the amount of HVJ-E employed in MRTs. Abstract: Mitochondrial replacement techniques (MRTs) provide a viable option for women carrying pathogenic mitochondrial DNA (mtDNA) variants to conceive disease-free offspring with a genetic connection. In comparison to electrofusion, HVJ-E-induced fusion has been identified as the most promising approach for clinical translation of MRTs due to its absence of electrical interference. However, despite confirmation of the absence of RNA activity in HVJ-E, a reduction in blastocyst quality has been observed in various MRTs studies utilizing the HVJ-E-induced fusion scheme. Recent investigations have revealed a dose-dependent elevation of reactive oxygen species (ROS) levels in various cancer cells incubated with HVJ-E. However, the impact of HVJ-E as a sole determinant on embryonic development in MRTs remains unverified. This investigation establishes that the augmented concentration of HVJ-E utilized in the conventional HVJ-E fusion protocol is an autonomous variable that influences embryonic development in MRTs. This effect may be attributed to amplified DNA damage resulting from heightened levels of ROS in reconstructed embryos. To mitigate the presence of HVJ-E in reconstructed zygotes while maintaining optimal fusion efficiency in MRTs, a novel HVJ-E-induced fusion approach was devised, namely, press-assisted fusion. This technique offers potential advantages in reducing detrimental factors that impede embryo development in MRTs.

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres.

RESEARCH QUESTION: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DESIGN: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. RESULTS: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. CONCLUSIONS: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

Optimal lead follicle size in letrozole human menopausal gonadotrophin intrauterine insemination cycles with and without spontaneous LH surge.

RESEARCH QUESTION: What is the optimal lead follicle size in letrozole, human menopausal gonadotrophin and intrauterine insemination (IUI) cycles with and without spontaneous LH surges? DESIGN: This retrospective cohort study included 3797 letrozole HMG IUI cycles between January 2010 and May 2021. All cycles were divided into two groups: the HCG trigger group (trigger day LH ≤15 mIU/ml) and the spontaneous LH surge group (trigger day LH >15 mIU/ml). These two groups were subdivided into smaller groups based on the diameter of the follicles. The primary outcome measure was clinical pregnancy rate. Logistic regression analysis was conducted to explore other risk factors. RESULTS: In the HCG trigger group, the clinical pregnancy rate varied significantly, with rates of 20.8%, 14.9% and 11.8% for the 16.1-18.0, 18.1-20.0 and 20.1-22.0 mm groups, respectively (P = 0.005). In the spontaneous LH surge group, the pregnancy rate of follicles within 14.1-16.0 mm was significantly higher than that of follicles within 20.1-22.0 mm (adjusted OR 0.533, 95% CI 0.308 to 0.923, P = 0.025). Also, patients with two lead follicles were 2.569 times more likely to achieve a clinical pregnancy than those with only one lead follicle (adjusted OR 2.569, 95% CI 1.258 to 5.246, P = 0.010). The duration of infertility was also found to be a common influencing factor in both groups. CONCLUSIONS: The optimal lead follicle size was between 16.1 and 18.0 mm in HCG-triggered letrozole HMG IUI cycles. If the lead follicle size is relatively small (14.1-18.0 mm) when a spontaneous LH surge occurs, there is no need to cancel the IUI cycle.

Impact of uterine malformations on pregnancy and neonatal outcomes of IVF/ICSI-frozen embryo transfer.

STUDY QUESTION: What is the impact of uterine malformations on reproductive and neonatal outcomes of IVF/ICSI-frozen embryo transfer? SUMMARY ANSWER: Unification defective uteri are associated with poorer neonatal outcomes including higher preterm delivery rate and lower birthweight, and septate uteri are associated with worse fertility outcomes including higher miscarriage and lower live birth rates (LBRs). WHAT IS KNOWN ALREADY: Several studies have investigated the negative effects of uterine malformations on pregnancy outcomes. However, an all-round and definitive conclusion has not been reached yet owing to the relatively low incidence of the disease and the heterogeneity of study populations, especially among women undergoing ART. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study including 411 women with congenital uterine anomalies and 14 936 women with a normal uterus who underwent first frozen-thawed embryo transfer cycles of IVF/ICSI from July 2008 to August 2019. We compared reproductive outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reproductive outcomes of women with uterine malformations were studied through three propensity score-matched comparisons (patients with bicorporeal uterus, septate uterus and hemi-uterus [bicorporeal, septate and hemi-uterus groups, respectively] along with corresponding control groups without uterine malformations). We also compared pregnancy and neonatal outcomes, and performed subgroup analysis addressing didelphus, bicornuate uteri and septate uteri before and after surgery independently. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to the matched control group, women with a bicorporeal uterus had a significantly lower LBR (24.4% versus 34.8%, odds ratio (OR) 0.61 [95% CI: 0.37, 1.00], P = 0.048). The incidence of miscarriage and preterm delivery was higher but not statistically significant (29.0% versus 18.1%, OR 1.85 [95% CI: 0.82, 4.19], P = 0.135; 22.6% versus 9.9%, OR 2.64 [95% CI: 1.07, 6.52], P = 0.063, respectively). In addition, the bicorporeal group had a significantly lower gestational age, higher caesarean rate and lower birthweight than bicorporeal control. Women with a septate uterus had comparable clinical pregnancy rates to controls (43.3% versus 49.9%, OR 0.77 [95% CI: 0.57, 1.04], P = 0.091), increased miscarriage rates (23.5% versus 13.0%, OR 2.05 [95% CI: 1.18, 3.58], P = 0.010) and lower LBRs (29.4% versus 42.2%, OR 0.57 [95% CI: 0.41, 0.79], P = 0.001). In both singleton and twins pregnancies, pregnancy and neonatal outcomes were comparable between women with a septate uterus and control. Women with a hemi-uterus had a tendency for lower clinical pregnancy rate (36.8% versus 42.3%, OR 0.80 [95% CI: 0.52, 1.21], P = 0.287) and LBR (29.8% versus 33.1%, OR 0.86 [95% CI: 0.55, 1.34], P = 0.502), compared to women without malformations. The incidences of miscarriage and preterm delivery, respectively, were 16.7% versus 16.6% (OR 1.01 [95% CI: 0.41, 2.47], P = 0.989), and 9.5% versus 11.4% (OR 0.82 [95% CI: 0.27, 2.51], P = 1) in women with a hemi-uterus as compared to control. LIMITATIONS, REASONS FOR CAUTION: This was a single-centre, retrospective study in which neonatal data were extracted from parental questionnaires. The information on uteri septum type and surgery methods was poorly presented, with limited detail. In patients with uterine malformations, the number of babies with birth defects and twin pregnancies was relatively small, limiting the power of the study. WIDER IMPLICATIONS OF THE FINDINGS: Compared to patients with a normal uterus, women with uterine malformation have poorer reproductive outcomes. Pregnant women with a uterine anomaly need to be managed as high-risk pregnancies and followed with appropriate obstetric review. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Ministry of Technology (2018YFC1003000), the Elite Team Project of Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JY201801), Shanghai Sailing Program (21YF1423200) and the Fundamental Research Program Funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ117). B.W.M. is supported by an NHMRC Investigatorgrant (GNT1176437). B.W.M. reports consultancy (with stock options) for ObsEva. B.W.M. has received research funding from Ferring and Merck. The authors declare no other competing interests. TRIAL REGISTRATION NUMBER: N/A.

Does the cell number of 0PN embryos on day 3 affect pregnancy and neonatal outcomes following single blastocyst transfer?

BACKGROUND: 0PN zygotes have a low cleavage rate, and the clinical outcomes of cleavage-stage embryo transfers are unsatisfactory. Blastocyst culturing is used to screen 0PN embryos, but whether the cell number of 0PN embryos on day 3 affects the clinical outcomes following single blastocyst transfer is unknown and would be helpful in evaluating the clinical value of these embryos. METHODS: This retrospective study compared 46,804 0PN zygotes, 242 0PN frozen-thawed single blastocyst transfers, and 92 corresponding 0PN singletons with 232,441 2PN zygotes, 3563 2PN frozen-thawed single blastocyst transfers, and 1250 2PN singletons from January 2015 to October 2019 at a tertiary-care academic medical centre. The 0PN and 2PN embryos were divided into two groups: the group with < 6 cells on day 3 and that with ≥ 6 cells. Embryo development, subsequent pregnancy and neonatal outcomes were compared between the two groups. RESULTS: The cleavage and available blastocyst rates of the 0PN zygotes were much lower than those of the 2PN zygotes (25.9% vs. 97.4%, P < 0.001; 13.9% vs. 23.4%, P < 0.001). In the < 6 cells group, the available blastocyst rate of the cleaved 0PN embryos was significantly lower than that of the 2PN embryos (2.5% vs. 12.7%, P < 0.001). However, in the ≥ 6 cells group, the available blastocyst rate of the 0PN cleaved embryos significantly improved, although it was slightly lower than that of the 2PN embryos (33.9% vs. 35.7%, P = 0.014). Importantly, compared to those of the 2PN single blastocyst transfers, the clinical pregnancy rate, live birth rate, Z-score and malformation rate of the 0PN single blastocyst transfers were not significantly different in either the < 6 cells group (30.4% vs. 39.8%, P = 0.362; 30.4% vs. 31.3%, P = 0.932; 0.89 ± 0.90 vs. 0.42 ± 1.02, P = 0.161; 0% vs. 2.6%, P = 1.000) or the ≥ 6 cells group (50.7% vs. 46.6%, P = 0.246; 39.7% vs. 38.3%, P = 0.677; 0.50 ± 1.23 vs. 0.47 ± 1.11, P = 0.861; 2.4% vs. 1.8%, P = 1.000). CONCLUSIONS: The cell number on day 3 of 0PN embryos affected the subsequent formation of blastocysts but did not influence the subsequent pregnancy and neonatal outcomes of 0PN single blastocyst transfers, which may be beneficial to clinicians counselling patients on the clinical value of 0PN embryos.

Live birth rate and neonatal outcomes following interventional embolization of hydrosalpinx.

BACKGROUND: Hydrosalpinx has a negative effect on the pregnancy outcomes of in vitro fertilization and embryo transfer (IVF-ET), and the pretreatment for hydrosalpinx play an important role in improving the outcomes of IVF-ET. This study aimed to investigate the impacts of interventional embolization of hydrosalpinx on the live birth rate and neonatal outcome after in-vitro fertilization. METHOD: In the present retrospective study, 3351 women receiving the first frozen embryo transfer (FET) after freeze-all policy were reviewed. Patients who received interventional embolization of hydrosalpinx (n = 1268) were included in the study group and those with hydrosalpinx-free bilateral fallopian tube obstruction (n = 2083) in the control group. The primary outcome was live birth (LB) rate; the secondary endpoints included rates of implantation, clinical pregnancy (CP), multiple pregnancy, and pregnancy loss. RESULTS: The LB rate was similar between embolization group (39.91%) and control group (43.21%) (P > 0.05). The rate of implantation (35.81% vs. 32.24%), CP (50.84% vs. 47%) and multiple pregnancy rate (28.71% vs. 24.16%) in the control group were significantly higher than in the embolization group (P < 0.05). The miscarriage rate (39.91%, vs 43.21%, P > 0.05), ectopic gestation rate (2.35% vs 2.83%, P > 0.05), and ongoing pregnancy rate (41.56% vs 44.89%, P > 0.05) were comparable between two groups. After adjustment for confounding factors, interventional embolization of hydrosalpinx was found to have no influence on the LB rate. The thicker endometrium, more embryos transferred, and transfer of blastocyst stage embryos significantly increased the LB rate and CP rate. CONCLUSION: The interventional embolization of hydrosalpinx can achieve the LB rate similar to that of hydrosalpinx-free obstruction patients with less risk, less pain and reduced medical cost. Thus, embolization of hydrosalpinx is one of the preferable clinical treatments for patients with hydrosalpinx.

The comparison of two different protocols ultra-long versus medroxyprogesterone acetate in women with ovarian endometriosis: a prospective randomized controlled trial.

BACKGROUND: This study aimed to investigate the medroxyprogesterone acetate (MPA) + HMG protocol vs ultra-long gonadotrophin releasing hormone (GnRH) agonist protocol in patients with advanced ovarian endometriosis who received in vitro fertilization (IVF). METHODS: Three hundred patients with advanced ovary endometriosis who underwent IVF were included, and embryological and clinical outcomes were assessed between March 2017 and September 2017. Patients were divided into MPA + HMG group and 1-month ultra-long GnRHa protocol group. RESULTS: Lower hMG dose and shorter medication time were found in the MPA + HMG group than in the GnRHa group (P < 0.05). Follicle to-Oocyte Index was significantly different between MPA + HMG group and GnRHa group (P < 0.001). No differences were found in the ovary response and numbers of mature oocytes, fertilized oocytes and viable embryos. The clinical pregnancy and live birth outcomes were similar between MPA + HMG group and GnRHa group, and these outcomes were independent of fresh or frozen embryo transfer in the GnRHa protocol group. There were no significant differences in the time to embryo transfer, medical cost and adverse effects. CONCLUSION: The number of oocytes retrieved and pregnancy outcomes after MPA + HMG protocol are similar to those after ultra-long GnRHa protocol in women with ovarian endometriosis. MPA + HMG protocol may be an alternative to ultra-long GnRHa protocol for IVF in ovary endometriosis patients. Trial registration The trial was registered in the Chinese Clinical Trial Registry (ChiCTR-INR-17010924) In conclusion, the administration of MPA in COH showed similar number of oocytes retrieved, no premature LH surge, and similar pregnancy and live birth outcomes in patients with advanced ovarian endometriosis undergoing IVF/ICSI as compared to the one-month long protocol. The use of MPA in COH appears to be promising although many questions remain to be elucidated, including the dose and time of progestin priming as well as its possible influence on the oocyte development potential and microenvironment. Given their good tolerability, few metabolic influence, and low cost, progestogens provide a novel alternative to the conventional protocol for patients with endometriosis.

Efficacy of artificial oocyte activation in patients with embryo developmental problems: a sibling oocyte control study.

PURPOSE: To explore whether artificial oocyte activation (AOA) can improve embryo developmental potentiality and pregnancy outcomes for patients with a history of embryo developmental problem. METHODS: This was a retrospective study and candidate patients with embryo development problems were collected. A total of 1422 MII eggs from the enrolled 140 patients were randomized divided equally into 2 groups, half for the AOA group (AOA), and the rest of sibling mature eggs for the control group (non-AOA). The patients were further divided into two subgroups: (1) the rate of good-quality day 3 embryos was 0% (group 1, n = 66); (2) the rate of good-quality day 3 embryos ranged from 1 to 30% (group 2, n = 74). RESULTS: In the early embryonic growth, there were no significant differences in the outcomes of AOA and non-AOA groups in terms of normal fertilization rates, cleavage rates, day 3 good-quality embryo rates and available blastocyst rates (72.7% vs. 79.3%, 97.4% vs. 98.0%, 20.1% vs. 19.7%, 6.6% vs. 8.4% in group 1, respectively; 77.7% vs. 81.9%, 98.1% vs. 97.0%, 25.8% vs. 22.1%, 9.6% vs. 9.3% in group 2, respectively). In the late embryonic growth, no significant differences were found in biochemical and clinical pregnancy rates, implantation rates, miscarriage rates, and live-birth rates (50.0% vs. 45.2%, 45.2% vs. 40.5%, 37.3% vs. 31.3%, 10.5% vs. 11.8%, 40.5% vs. 35.7%, respectively) between two groups. In addition, neonatal outcomes were similar in both the groups as well. CONCLUSION: Our study demonstrated that the AOA using ionomycin 1 h after ICSI did not bring benefits to the early or late development of embryos derived from patients with a history of embryo developmental problems.

Ptk2b deletion improves mice folliculogenesis and fecundity via inhibiting follicle loss mediated by Erk pathway.

Ptk2b has been found playing critical roles in oocyte maturation and subsequent fertilization in vitro. But what is the exact in vivo function in reproduction still elusive. Here, by constructing Ptk2b mutant mice, we found Ptk2b was not essential for mice fertility, unexpectedly, contrary to previously reported in vitro findings, we found Ptk2b ablation significantly improved female fecundity. Follicle counting indicated that the number of primordial follicles and growing follicles in matured mice was significantly increased in the absence of Ptk2b, whereas the primordial follicle formation showed no defects. We also found this regulation was in an autophosphorylation independent pathway, as autophosphorylation site mutant mice (PTK2BY402F ) show no phenotype in female fertility. Further biochemistry studies revealed that Ptk2b ablation promotes folliculogenesis via Erk pathway mediate follicle survival. Together, we found a novel biological function of Ptk2b in folliculogenesis, which could be potentially used as a therapeutic target for corresponding infertility.

Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility.

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted in vivo. In clinical practice, some infertility case subjects suffer from recurrent failure of in vitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein in vitro and in affected individuals' oocytes in vivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 in vitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts in vitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.

Monolithic integration of ultraviolet light emitting diodes and photodetectors on a p-GaN/AlGaN/GaN/Si platform.

In this letter, we report the first demonstration of monolithically integrated ultraviolet (UV) light emitting diodes (LEDs) and visible-blind UV photodetectors (PDs) employing the same p-GaN/AlGaN/GaN epi-structures grown on Si. Due to the radiative recombination of holes from the p-GaN layer with electrons from the 2-D electron gas (2DEG) accumulating at the AlGaN/GaN heterointerface, the forward biased LED with p-GaN/AlGaN/GaN junction exhibits uniform light emission at 360 nm. Facilitated by the high-mobility 2DEG channel governed by a p-GaN optical gate, the visible-blind phototransistor-type PDs show a low dark current of ∼10-7 mA/mm and a high responsivity of 3.5×105 A/W. Consequently, high-sensitivity photo response with a large photo-to-dark current ratio of over 106 and a response time less than 0.5 s is achieved in the PD under the UV illumination from the on-chip adjacent LED. The demonstrated simple integration scheme of high-performance UV PDs and LEDs shows great potential for various applications such as compact opto-isolators.

Advanced trophectoderm quality increases the risk of a large for gestational age baby in single frozen-thawed blastocyst transfer cycles.

STUDY QUESTION: Does trophectoderm (TE) quality affect birthweight after single frozen-thawed blastocyst transfer? SUMMARY ANSWER: Transfer of single blastocyst with advanced TE quality was associated with higher birthweight and increased risk of a large for gestational age (LGA) baby. WHAT IS KNOWN ALREADY: Transfer of blastocysts with advanced TE quality results in higher ongoing pregnancy rates and a lower miscarriage risk. However, data on the relationship between TE quality and birthweight are still lacking. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study at a tertiary-care academic medical center included 1548 singleton babies born from single frozen-thawed blastocyst transfer from January 2011 to June 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Babies were grouped into four groups according to embryo expansion (Stages 3, 4, 5 and 6), three groups according to inner cell mass (ICM) quality (A, B and C), and three groups according to TE quality (A, B and C). Main outcomes included absolute birthweight, Z-scores adjusted for gestational age and gender, and adverse neonatal outcomes. Multivariable linear and logistic regression analyses were performed to investigate the association of neonatal outcomes with expansion stage, ICM quality and TE quality. MAIN RESULTS AND THE ROLE OF CHANCE: As TE quality decreased, birthweight (3468.10 ± 471.52, 3357.69 ± 522.06, and 3288.79 ± 501.90 for A, B and C, respectively, P = 0.002), Z-scores (0.59 ± 1.07, 0.42 ± 1.04, and 0.27 ± 1.06 for A, B and C, respectively, P = 0.002) and incidence of LGA (28.9%, 19.7% and 17.4% for A, B and C, respectively, P = 0.027) decreased correspondingly. After adjusting for confounders, compared with the Grade A group, blastocysts with TE Grade B (standardized coefficients (ß): -127.97 g, 95% CI: -234.46 to -21.47, P = 0.019) and blastocysts with TE grade C (ß: -200.27 g, 95% CI: -320.69 to -79.86, P = 0.001) resulted in offspring with lower birthweight. Blastocysts with TE grade C brought babies with lower Z-scores than TE Grade A (ß: -0.35, 95% CI: -0.59 to -0.10, P = 0.005). Also, embryos with TE Grade B (adjusted odds ratio (aOR):0.91, 95% CI: 0.84 to 0.99, P = 0.033) and embryos with TE Grade C (aOR : 0.89, 95% CI: 0.81 to 0.98, P = 0.016) had lower chance of leading to a LGA baby than those with TE Grade A. No association between neonatal outcomes with embryo expansion stage and ICM was observed (all P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The retrospective design, lack of controlling for several unknown confounders, and inter-observer variation limited this study. WIDER IMPLICATIONS OF THE FINDINGS: The study extends our knowledge of the down-stream effect of TE quality on newborn birthweight and the risk of LGA. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by National Key R&D Program of China (2018YFC1003000), National Natural Science Foundation of China (81771533 to Y.P.K. and 31200825 to L.S.) and Innovative Research Team of High-level Local Universities in Shanghai (SSMU-ZLCX20180401), Shanghai Sailing Program(21YF1423200) and the Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (JYZZ117). The authors declare no conflict of interest in this present study. TRIAL REGISTRATION NUMBER: N/A.

DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation.

BACKGROUND: The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages. METHODS: We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer. RESULTS: The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls. CONCLUSIONS: AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.

Quercetin prevents primordial follicle loss via suppression of PI3K/Akt/Foxo3a pathway activation in cyclophosphamide-treated mice.

BACKGROUND: Chemotherapy improves the survival rates of patients with various cancers but often causes some adverse effects, including ovarian damage, characterised by a decrease in primordial follicle stockpiles. Recent studies have revealed that chemotherapy may stimulate the PI3K signalling pathway, thereby resulting in accelerated primordial follicle activation and a decreased ovarian reserve. Quercetin is an inhibitor of the PI3K pathway; however, its protective effects against chemotherapy-induced follicle loss in mice have not been established. In this study, the effects of quercetin in a mouse model of cyclophosphamide-induced ovarian dysfunction were investigated. METHODS: C57BL/6 female mice were used for the study. Paraffin sections of mouse ovaries (n = 30 mice) were stained with haematoxylin and eosin for differential follicle counts. Apoptosis (n = 5 mice per group) was evaluated by TUNEL assay. Immunohistochemical staining for ki67 and Foxo3a (n = 5 mice per group) was performed to evaluate the activation of primordial follicles. The role of the PI3K signalling pathway in the ovaries (n = 45 mice) was assessed by western blotting. RESULTS: Quercetin attenuated the cyclophosphamide-induced reduction in dormant primordial follicles. Analysis of the PI3K/Akt/Foxo3a pathway showed that quercetin decreased the phosphorylation of proteins that stimulate follicle activation in cyclophosphamide-induced ovaries. Furthermore, quercetin prevented cyclophosphamide-induced apoptosis in early growing follicles and early antral follicles, maintained anti-Müllerian hormone levels secreted by these follicles, and preserved the quiescence of the primordial follicle pool, as determined by intranuclear Foxo3a staining. CONCLUSIONS: Quercetin attenuates cyclophosphamide-induced follicle loss by preventing the phosphorylation of PI3K/Akt/Foxo3a pathway members and maintaining the anti-Müllerian hormone level through reduced apoptosis in growing follicles. Accordingly, quercetin is expected to improve fertility preservation and the prevention of endocrine-related side effects of chemotherapy.

YTHDF2 is essential for spermatogenesis and fertility by mediating a wave of transcriptional transition in spermatogenic cells.

The dynamic and reversible regulation roles of m6A modification and the characterization of m6A readers have provided new insights into spermatogenesis at the post-transcriptional level. YTHDF2, as an m6A reader, has been reported to mediate the m6A-containing transcript decay during the mouse oocyte maturation, embryonic stem cell differentiation, neural development, and zebrafish maternal-to-zygotic transition. However, the roles of YTHDF2 in mammalian spermatogenesis are uncertain. Here, we generated germ cell-specific Ythdf2 mutants (Ythdf2-vKO) at a C57BL/6J background and demonstrated that YTHDF2 is essential for mouse spermatogenesis and fertility. Ythdf2-vKO provides oligoasthenoteratozoospermia phenotype with increased apoptosis in germ cells. High-throughput RNA-seq analysis showed that a group of mRNAs is upregulated in Ythdf2-vKO mouse testis; further analysis and MeRIP-qPCR data showed that most of the upregulated genes in Ythdf2-vKO mouse testis are modified with m6A and are YTHDF2 candidate binding genes. Interestingly, RNA-seq analysis combined with our previous single-cell transcriptomics data of mouse spermatogenesis pointed out the failure of a wave of transcript transition during the spermatogenesis of Ythdf2-vKO mice, which was confirmed by gene expression analysis using qPCR of diplotene spermatocytes and round spermatids obtained through fluorescence-activated cell sorting. Our study demonstrates the fundamental role of YTHDF2 during mouse spermatogenesis and provides a potential candidate for the diagnosis of male infertility with the oligoasthenoteratozoospermia syndrome.

Ionomycin-induced mouse oocyte activation can disrupt preimplantation embryo development through increased reactive oxygen species reaction and DNA damage.

Oocyte activation induced by calcium oscillations is an important process in normal fertilization and subsequent embryogenesis. In the clinical-assisted reproduction, artificial oocyte activation (AOA) is an effective method to improve the clinical outcome of patients with null or low fertilization rate after ICSI. However, little is known about the effect of AOA on preimplantation embryo development in cases with normal fertilization by ICSI. Here, we used ionomycin at different concentrations to activate oocytes after ICSI with normal sperm and evaluated energy metabolism and preimplantation embryo development. We found that a high concentration of ionomycin increased the frequency and amplitude of calcium oscillation patterns, affecting the balance of mitochondrial energy metabolism, leading to increased reactive oxygen species (ROS) and decreased ATP. Eventually, it increases DNA damage and decreases blastocyst formation. In addition, the addition of vitamin C to the culture medium ameliorated the increase in ROS and DNA damage and rescued the abnormal embryo development caused by excessive ionomycin activation. This study provides a perspective that the improper application of AOA may have adverse effects on preimplantation embryo development. Thus, clinical AOA treatment should be cautiously administered.

Association between peak serum estradiol level during controlled ovarian stimulation and neonatal birthweight in freeze-all cycles: a retrospective study of 8501 singleton live births.

STUDY QUESTION: Is there an association between peak serum estradiol (E2) level during controlled ovarian stimulation (COS) and neonatal birthweight in freeze-all cycles? SUMMARY ANSWER: Peak serum E2 level during ovarian stimulation is not associated with neonatal birthweight in freeze-all cycles. WHAT IS KNOWN ALREADY: Supraphysiologic E2 levels during COS have been demonstrated to generate a suboptimal peri-implantation endometrial environment and thus lead to adverse neonatal outcomes in fresh embryo transfer cycles. Previous experimental studies also suggested a potential influence of superovulation on oocyte epigenetic programming, but whether it translates into altered phenotypes of fetal growth and development remains unclear in clinical practice. By segmenting the process of COS and embryo transfer, the freeze-all policy provides a novel model to investigate the sole impact of ovarian stimulation on oocytes after ruling out the effects of hyperestrogenic milieu on endometrium in fresh cycles. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of 8501 patients who underwent their first COS cycles with a freeze-all strategy and delivered live-born singletons in subsequent frozen-thawed embryo transfer cycles from January 2007 to December 2016 at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were categorized into six groups according to E2 level on trigger day in regular increments of 1000 pg/mL: <1000, 1000-1999, 2000-2999, 3000-3999, 4000-4999 and ≥5000 pg/mL. Univariable and multivariable linear regression and logistic regression analysis were performed to assess the independent association between peak E2 level and measures of neonatal birthweight including absolute birthweight, Z-score, low birthweight (LBW) and small-for-gestational age (SGA). MAIN RESULTS AND THE ROLE OF CHANCE: The six groups did not differ significantly in birthweight, Z-score or the incidence of LBW and SGA. Compared with the E2 <1000 pg/mL group, the adjusted mean difference (95% confidence interval [CI]) of stratified higher E2 groups was 17.2 (-31.0-65.5), 12.3 (-35.9-60.5), -4.1 (-51.9-43.7), -0.6 (-48.9-47.8) and -3.6 (-50.0-42.8) g for birthweight, and 0 (-0.11-0.10), 0.02 (-0.08-0.12), 0.04 (-0.06-0.14), -0.01 (-0.11-0.10) and -0.04 (-0.14-0.06) for Z-score, respectively. Regarding the outcomes of LBW and SGA, no increased risks were observed in each E2 category, with the adjusted odds ratio (95% CI) being 1.21 (0.68-2.16), 1.0 (0.58-1.90), 0.90 (0.50-1.63), 0.93 (0.51-1.69) and 1.08 (0.61-1.90) for LBW, and 0.97 (0.58-1.64), 1.06 (0.63-1.77), 0.77 (0.46-1.31), 0.71 (0.41-1.22) and 1.00 (0.60-1.65) for SGA, respectively. LIMITATIONS, REASONS FOR CAUTION: The study was retrospective in design, and other unknown confounding factors may not be included for adjustment. Furthermore, the generalization of the study finding could be limited to some extent by the majority of double cleavage-stage embryo transfer and difference in birthweight reference percentiles between Chinese and other populations. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that the hyperestrogenic milieu during COS does not seem to pose adverse effects on neonatal birthweight after frozen-thawed embryo transfer, which provides reassuring information for high ovarian responders in freeze-all cycles concerning their offspring's health. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the National Key Research and Development Program of China (SQ2018YFC100163) and National Natural Science Foundation of China (81571397, 81771533). The authors declare no conflict of interest.

The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.

STUDY QUESTION: To evaluate the impact of storage time after vitrification on embryo viability, pregnancy outcomes and neonatal outcomes. SUMMARY ANSWER: The prolonged storage time of vitrified embryos negatively affected pregnancy outcomes, including biochemical pregnancy rate, clinical pregnancy and live birth rate; but did not influence neonatal outcomes. WHAT IS KNOWN ALREADY: Although vitrification has been the fundamental tool of ART treatments in recent years, few studies have explored the influence of storage period after vitrification on embryonic and clinical outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed among 24 698 patients with the first vitrified embryo transfer following a freeze-all strategy during the period from January 2011 to December 2017. PARTICIPANTS/MATERIAL, SETTING, METHODS: A total of 24 698 patients met the inclusion criteria and were grouped according to the storage time (11 330 patients in Group 1 with storage time <3 months, 9614 patients in Group 2 with storage time between 3 and 6 months, 3188 patients in Group 3 with storage time between 6 and 12 months and 566 in Group 4 with storage time between 12 and 24 months). The pregnancy outcomes and neonatal outcomes were compared between different storage time groups. Multivariate logistic regression and linear regression were performed to evaluate the independent effect of storage time on clinical outcomes, adjusting for important confounders. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for potential confounding factors, the chance of biochemical pregnancy (Group 1 as reference; Group 2: adjusted odds ratio (aOR) = 0.92, 95% CI 0.87-0.97; Group 3: aOR = 0.83, 95% CI 0.76-0.90; Group 4: aOR = 0.68, 95% CI 0.56-0.81), clinical pregnancy (Group 2: aOR = 0.91, 95% CI 0.86-0.96; Group 3: aOR = 0.80, 95% CI 0.73-0.87; Group 4: aOR = 0.65, 95% CI 0.54-0.79) and live birth (Group 2: aOR = 0.89, 95% CI 0.85-0.95; Group 3: aOR = 0.83, 95% CI 0.76-0.91; Group 4: aOR = 0.59, 95% CI 0.48-0.72) significantly decreased with the increasing storage time, whereas the relationship between miscarriage, ectopic pregnancy and storage time did not reach statistical significance. In addition, there was no evidence of differences in adverse neonatal outcomes (preterm birth, low birthweight, high birthweight, macrosomia or birth defects) between groups. LIMITATION, REASONS FOR CAUTION: Our study was limited by the retrospective design from a single center, the conclusion from our study needs to be verified in further studies. WIDER IMPLICATIONS OF THE FINDINGS: This study provides new findings about the relationship between prolonged storage time of vitrified embryos and clinical outcomes and offers evidence for the safety of using long-stored embryos after vitrification. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (grant nos. 81903324, 81771533, 81571397, 81701523), National Key Research and Development Program of China (grant no. SQ2018YFC100163). None of the authors have any conflicts of interest to declare.

Novel mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization.

STUDY QUESTION: Do sperm-specific phospholipase C zeta (PLCZ1) mutations account for male infertility due to fertilization failure? SUMMARY ANSWER: Six novel mutations and one reported mutation in PLCZ1 were identified in five of 14 independent families characterized by fertilization failure or poor fertilization, suggesting that these mutations may be responsible for fertilization failure in men exhibiting primary infertility. WHAT IS KNOWN ALREADY: PLCZ1 is essential for the induction of intracellular calcium (Ca2+) oscillations and the initiation of oocyte activation during mammalian fertilization. However, genetic evidence linking PLCZ1 mutations with male infertility remains limited. STUDY DESIGN, SIZE, DURATION: Fourteen unrelated primary infertility patients were recruited into this study from January 2016 to December 2018; the patients exhibited total fertilization failure or poor fertilization, as evidenced by ICSI and sperm-related oocyte activation deficiencies identified in mouse oocyte activation assays. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genomic DNA samples were extracted from the peripheral blood of patients. The whole exons of PLCZ1 were sequenced by Sanger sequencing. The PLCZ1 sequences were aligned by CodonCode software to identify rare variants. The ExAC database was used to search for the frequency of corresponding mutations. The pathogenicity of identified variants and their possible effects on the protein were assessed in silico. PLCZ1 protein levels in semen samples were evaluated by western blotting. Oocyte activation ability was assessed by the injection of wild-type and mutant PLCZ1 cRNAs into human mature metaphase II (MII) oocytes in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: We identified six novel mutations and one reported mutation in PLCZ1 among five affected individuals. In addition to four novel missense mutations, two new types of genetic variants were identified, including one in-frame deletion and one splicing mutation. Western blot analysis revealed that PLCZ1 protein expression was not observed in the semen samples from the five affected patients. Microinjection with the PLCZ1 cRNA variants was performed, and a significant decrease in the percentage of pronuclei was observed for four novel missense mutations and one novel in-frame deletion mutation, suggesting that these mutations have a deleterious influence on protein function. By artificial oocyte activation treatment, the fertilization failure phenotypes of four affected patients were successfully rescued and three healthy babies were delivered. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We screened only the whole exons of PLCZ1. Additional possible mutations in the non-coding region of PLCZ1 should be further studied. WIDER IMPLICATIONS OF THE FINDINGS: Our study not only further confirms the important role of PLCZ1 in human fertilization but also expands the mutational spectrum of PLCZ1 associated with male infertility, which provides a basis for assessing genetic variation in PLCZ1 as a potential diagnostic marker for infertile men suffering from fertilization failure. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Foundation of China (81 571 486 and 81 771 649). All authors have no conflicts of interest to declare.