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.

Earlier second polar body transfer and further mitochondrial carryover removal for potential mitochondrial replacement therapy.

The second polar body (PB2) transfer in assisted reproductive technology is regarded as the most promising mitochondrial replacement scheme for preventing the mitochondrial disease inheritance owing to its less mitochondrial carryover and stronger operability. However, the mitochondrial carryover was still detectable in the reconstructed oocyte in conventional second polar body transfer scheme. Moreover, the delayed operating time would increase the second polar body DNA damage. In this study, we established a spindle-protrusion-retained second polar body separation technique, which allowed us to perform earlier second polar body transfer to avoid DNA damage accumulation. We could also locate the fusion site after the transfer through the spindle protrusion. Then, we further eliminated the mitochondrial carryover in the reconstructed oocytes through a physically based residue removal method. The results showed that our scheme could produce a nearly normal proportion of normal-karyotype blastocysts with further reduced mitochondrial carryover, both in mice and humans. Additionally, we also obtained mouse embryonic stem cells and healthy live-born mice with almost undetectable mitochondrial carryover. These findings indicate that our improvement in the second polar body transfer is conducive to the development and further mitochondria carryover elimination of reconstructed embryos, which provides a valuable choice for future clinical applications of mitochondrial replacement.

Prediction of live birth in vitrified-warmed 1PN-derived blastocyst transfer: Overall quality grade, ICM, TE, and expansion degree.

Background: Numerous studies have reported that transfer of blastocysts derived from monopronuclear (1PN) zygotes achieved live births. However, the potential value of morphology grading for the prediction of 1PN blastocyst viability is unclear, and the blastocyst selection criterion for successful pregnancy has not been set up yet. The aim of this study is to assess the ability of the blastocyst morphology grading system based on three parameters, namely, inner cell mass (ICM), trophectoderm (TE), and expansion degree and to predict outcomes of a cycle with single 1PN blastocyst transfer. Methods: A total of 266 vitrified-warmed 1PN-derived blastocyst transfer cycles for IVF treatment at Shanghai Ninth People's Hospital between 2007 and 2020 were included. The study was performed on single blastocyst transfers. Electronic records of patients were retrospectively analyzed. In the current study, the blastocysts were classified into three groups: "good," 3-6AA, 3-6AB, 3-6BA; "medium," 3-6BB, 3-6AC, 3-6CA; and "poor," 3-6BC, 3-6CB, 3-6CC. The basal characteristics, embryo grading, and clinical outcomes were compared between the three groups. The association of morphology parameters with pregnancies and live births was analyzed. Logistic regression was adopted to set up a prediction model of live births. Results: Transfer of the good-quality blastocysts achieved significant higher pregnancies (biochemical pregnancy: 59%; clinical pregnancy: 56.4%, and live birth 48.7%) than those in the group of the medium (biochemical pregnancy: 59%; clinical pregnancy: 49.6%; live birth: 40.4%) or poor-quality (biochemical pregnancy: 38.4%; clinical pregnancy: 34.9%; live birth: 26.7%) blastocysts (p < 0.05). There was a significant association between ICM and live birth. A prediction model of live births involving ICM, TE, and expansion degree was set up. Conclusion: In 1PN transfer cycles, a higher overall blastocyst quality is shown to correlate most strongly with optimal pregnancy and live birth outcomes. The selection of high-quality blastocysts for transfer should consider the ICM score first. The prediction model of live births based on ICM, TE, and expansion degree may help predict successful pregnancy in 1PN single-blastocyst transfer cycles.

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.

Luteinizing Hormone Suppression by Progestin-Primed Ovarian Stimulation Is Associated With Higher Implantation Rate for Patients With Polycystic Ovary Syndrome Who Underwent in vitro Fertilization/Intracytoplasmic Sperm Injection Cycles: Comparing With Short Protocol.

BACKGROUND: Many studies have demonstrated the positive clinical value of progestin-primed ovarian stimulation (PPOS) in patients with polycystic ovary syndrome (PCOS) who underwent assisted reproductive technology. However, the underlying factors contributing to this phenomenon remain unclear. We conducted a retrospective observational study to compare the clinical outcomes of women with PCOS who underwent PPOS or the short protocol to identify possible factors that influence the outcome. METHODS: This study included 304 patients who underwent PPOS and 152 patients who underwent short protocol from April 2014 to July 2019 after propensity-score matching. Human menopausal gonadotropin (hMG) dose, hormone profile, embryo development, and clinical outcomes of frozen-thawed embryo transfer (FET) cycles were compared. The primary outcome measure was the implantation rate. Logistic regression was performed to identify contributing factors, and receiver operating characteristic curve analysis was used to calculate the cutoff of luteinizing hormone (LH) difference ratio in clinical outcomes. RESULTS: Compared with the short protocol, PPOS resulted in a higher implantation rate (43.4% vs. 31.9%, P < 0.05), clinical pregnancy rate (61.8% vs. 47.4%, P < 0.05), and live birth rate (48.4% vs. 36.8%, P < 0.05). Similar fertilization, cleavage, and valid embryo rate per oocyte retrieved between groups were observed. The LH difference ratio was positively associated with implantation rate [P = 0.027, odds ratio (OR) = 1.861, 95% CI: 1.074-3.226]. The relationship between the LH difference ratio with clinical outcomes was confirmed by receiver operating characteristic curve analysis and comparisons among patients grouped by the LH difference ratio. CONCLUSION: The implantation rate was associated with the LH difference ratio during ovary stimulation in patients with PCOS. Our results provide the explanation why PPOS shows the positive clinical outcomes for patients with PCOS.

The Valuable Reference of Live Birth Rate in the Single Vitrified-Warmed BB/BC/CB Blastocyst Transfer: The Cleavage-Stage Embryo Quality and Embryo Development Speed.

BACKGROUND: It is unclear whether we should focus attention on cleavage-stage embryo quality and embryo development speed when transferring single particular grade vitrified-warmed blastocysts, especially poor-quality blastocysts (grade "C"). METHOD: This retrospective study considered 3386 single vitrified-warmed blastocyst transfer cycles from January 2010 to December 2017. They were divided into group 1 (AA/AB/BA, n = 374), group 2 (BB, n = 1789), group 3 (BC, n = 901), and group 4 (CB, n = 322). The effects of cleavage-stage embryo quality and embryo development speed were measured in terms of clinical pregnancy and live birth rates in each group. RESULTS: Pregnancy outcomes showed a worsening trend from groups 1 to 4; the proportion of embryos with better cleavage-stage quality and faster development speed decreased. In group 1, only the blastocyst expansion degree 3 was a negative factor in the clinical pregnancy rate (odds ratio (OR) [95% confidence interval (CI)]: 0.233 [0.091-0.595]) and live birth rate (0.280 [0.093-0.884]). In the other groups (BB, BC, and CB), blastocysts frozen on day 5 had significantly better clinical pregnancy outcomes than those frozen on day 6: 1.373 [1.095-1.722] for group 2, 1.523 [1.055-2.197] for group 3, and 3.627 [1.715-7.671] for group 4. The live birth rate was 1.342 [1.060-1.700] for group 2, 1.544 [1.058-2.253] in group 3, and 3.202 [1.509-6.795] in group 4, all Ps < 0.05). The degree of blastocoel expansion three for clinical pregnancy rate in group 2 (0.350 [0.135-0.906], P < 0.05) and day 3 blastomere number (>7) for live birth rate in group 4 (2.455 [1.190-5.063], P < 0.05) were two important factors. CONCLUSION: We should consider choosing BB/BC/CB grade blastocysts frozen on day 5, CB grade blastocysts with day 3 blastomere numbers (>7), and AA/AB/BA grade blastocysts with degrees of expansion (≥4) to obtain better pregnancy outcomes.

Effect of endometrial thickness on birthweight in frozen embryo transfer cycles: an analysis including 6181 singleton newborns.

STUDY QUESTION: Does endometrial thickness (EMT) have an impact on singleton birthweight in frozen embryo transfer (FET) cycles? SUMMARY ANSWER: An EMT <8 mm was associated with a lower mean birthweight and gestational age- and gender-adjusted birthweight (Z-scores) of singletons resulting from FET. WHAT IS KNOWN ALREADY: Previous studies have examined the impact of EMT on IVF success rates. Little is known, however, regarding the relationship between EMT and neonatal birthweight. STUDY DESIGN, SIZE, DURATION: This retrospective study involved singleton live births born to women undergoing frozen-thawed Day 3 embryo transfer during the period from January 2010 to December 2017 at a tertiary care centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 6181 women who fulfilled the inclusion criteria were included and were grouped into five groups depending on the EMT: <8 mm, 8-9.9 mm, 10-11.9 mm, 12-13.9 mm and ≥14 mm. EMT between 10 and 11.9 mm was taken as a reference group. Singleton birthweight was the primary outcome measure. A multivariable linear regression analysis was performed to detect a relationship between EMT and newborns' birthweight after controlling for a number of potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: A modest but significant decrease in birthweight was observed in the EMT <8 mm group as compared with groups with EMT ≥10 mm, with a mean difference of 89-108 g. Also, singletons from the EMT <8 mm group (0.24 ± 1.04) had a significantly lower birthweight Z-scores than those from the EMT 10-11.9 mm (0.41 ± 1.02; P = 0.032) or EMT 12-13.9 mm (0.46 ± 1.07; P = 0.004) groups. Further, multiple linear regression analyses indicated that parental BMIs, gestational age, newborn gender, pregnancy complications and EMT <8 mm were all independent predictors of neonatal birthweight. LIMITATIONS, REASONS FOR CAUTION: The present study was limited by its retrospective design. Future prospective studies are required to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provided new insight into the relationship between EMT and neonatal outcomes by showing that a thin endometrium is associated with a decrease in singleton birthweight. STUDY FUNDING/COMPETING INTEREST(S): National Key Research and Development Program of China (2018YFC1003000); the National Natural Science Foundation of China (81771533, 81571397, 31770989, 81671520); the China Postdoctoral Science Foundation (2018M630456). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Obstetrical and neonatal outcomes after transfer of cleavage-stage and blastocyst-stage embryos derived from monopronuclear zygotes: a retrospective cohort study.

OBJECTIVE: To evaluate the outcomes of embryo transfer with the use of monopronuclear (1PN) zygotes, and the risks of congenital malformations and postpartum developmental disorders. DESIGN: Retrospective cohort study. SETTING: Tertiary-care academic medical center. PATIENT(S): This study included patients who underwent single embryo transfer of 1PN frozen-thawed cleavage- or blastocyst-stage embryos. Seventy-six singletons were assessed for congenital malformations and defects in psychomotor development. INTERVENTION(S): Monopronuclear frozen-thawed cleavage-stage or blastocyst embryos were compared with 2PN frozen-thawed cleavage-stage and blastocyst frozen embryos, in frozen-thawed embryo transfer (FET) cycles. Neonates from 2PN FET constituted the control group. MAIN OUTCOME MEASURE(S): Implantation rate (IR), clinical pregnancy rate (PR), miscarriage rate, live birth rate, congenital malformations, and motor and language development status were compared. RESULT(S): The cohort comprised 186, 676, 133, and 502 patients consenting to FET with, respectively, 1PN cleavage-stage, 2PN cleavage-stage, 1PN blastocystocyst, and 2PN blastocystocyst embryos. The IR, PR, and live birth rate were lower in 1PN than with 2PN cleavage-stage FET, but similar between 1PN and 2PN blastocystocysts. Miscarriage rates did not differ significantly between 1PN and 2PN cleavage-stage or blastocystocyst FET. Risk of congenital malformations and defects in psychomotor development did not differ significantly within 2 years postpartum with the use of 1PN or 2PN FET. CONCLUSION(S): The present results suggest that the value of 1PN blastocyst FET is similar to that of 2PN blastocyst FET, without an increased risk of miscarriage rate, congenital malformations, or defects of psychomotor development.

Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level.

Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5' splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads.