Sex ratio shift after frozen single blastocyst transfer in relation to blastocyst morphology parameters.

The sex ratio shift was observed in peoples who underwent ART treatment. Moreover, there is limited evidence on differences in sex ratio between single frozen-thawed blastocyst morphology, insemination type and transfer days. So further research is needed in this area with regard to factors possibly affecting the sex ratio. Retrospective study based on multicenter including two large assisted reproduction centers in Shanghai and Wuhan in China. A total of 6361 singleton delivery offspring after frozen-thawed blastocyst transfer. Propensity score weighting and logistic regression models were used to estimate the associations between blastocyst morphology grading and child sex ratio. The main outcome measures is singleton sex ratio. In our study, the primary outcome measure was sex ratio which was calculated as the proportion of male newborns among all live births. Higher quality blastocysts resulted in a higher sex ratio than single poor-quality frozen-thawed blastocyst transfer. Among the three blastocyst morphological parameters of trophectoderm (TE), Grade A and B were significantly associated with a higher sex ratio than Grade C. The similar trend was observed in both IVF and ICSI treated subgroups. As compared with expansion (4 + 3), expansion degree 6 achieved a higher sex ratio in overall populations and IVF treated subgroup. Transferring blastocysts of day 6 had the highest sex ratio both in IVF group and ICSI group. A 6.95% higher sex ratio in transferring blastocysts of day 5 in IVF group than those in ICSI group. No significant association between inner cell mass degree and sex ratio was observed. However, as compared with IVF treatment, all morphology parameters achieved the similar or the biased sex ratio favoring female in ICSI treated subgroup. Quality of blastocysts was positively associated with sex ratio. TE score and expansion degree rather than ICM were significantly associated with sex ratio at birth. ICSI treatment promotes the biased sex ratio favoring female.

Novel WEE2 homozygous mutations c.1346C>T and c.949A>T identified in primary infertile women due to unexplained fertilization failure.

The occurrence of unexplained fertilization failure can have profound psychological and financial consequences for couples struggling with infertility, and its pathogenesis remains unclear. Increasing evidence highlights genetic basis of unexplained fertilization failure occurrence. Here, we identified one novel homozygous nonsense mutation (c.949A>T), one novel homozygous missense mutation (c.1346C>T), and three reported homozygous mutations (c.585G>C, c.1006_1007insTA, c.1221G>A) in six unrelated probands, showing similar manifestations of unexplained fertilization failure. This finding expands the spectrum of WEE2 mutations, highlighting the critical role of WEE2 in fertilization process, and provides a basis for the prognostic value of testing for WEE2 mutations in primary infertile couples with unexplained fertilization failure.

COVID-19 infection after oocyte retrieval did not have detrimental effects on embryo implantation for frozen embryo transfer.

Patients preparing for their renewal fertility treatments with embryos frozen before coronavirus disease 2019 (COVID-19) infection do not need to be concerned about the potential impact of COVID-19 infection on oocyte quality and embryonic development. However, many women are still hesitant to undergo frozen embryo transfer (FET) due to fear of the detrimental effect of COVID-19 infection on endometrial receptivity and embryo implantation. The objective was to explore whether COVID-19 infection after oocyte retrieval is related to an increased risk of adverse pregnancy outcomes in a cohort of Chinese women undergoing FET. A retrospective cohort study was conducted among 300 infertile women undergoing FET with embryos frozen before COVID-19 infection. Women were categorized into noninfection, infection before FET, or infection after FET groups. Multivariable logistic regression was performed to assess the association of COVID-19 infection with clinical pregnancy outcomes, including biochemical pregnancy, clinical pregnancy, and early miscarriage. The implantation rates for patients in the group with infection before FET (29.14%) and the group with infection after FET (30.38%) were not significantly lower than those in the noninfection group (31.03%). The rate of biochemical pregnancy (54.55% vs. 52.27%, p = 0.750; 43.14% vs. 52.27%, p = 0.209) was not significantly different among the three groups. Although the clinical pregnancy rate showed a declining trend from 45.45% in the noninfection group to 38.27% in the group with infection after FET, this result was not statistically significant. The early miscarriage rate was similar in the group with infection before FET and the group with infection after FET compared with that in the noninfection group (3.64% vs. 5.68%, p = 0.496; 6.86% vs. 5.68%, p = 0.739). After adjusting for potential confounders, the biochemical pregnancy rate, clinical pregnancy rate, and early miscarriage rate were not significantly different for patients with infection before or after FET compared with patients without infection. This research indicated that COVID-19 infection after oocyte retrieval with embryos frozen before infection did not cause any detrimental effect on endometrial receptivity for embryo implantation.

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.

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.

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.

Progesterone affects clinic oocyte yields by coordinating with follicle stimulating hormone via PI3K/AKT and MAPK pathways.

Introduction: As an effective inhibitor of premature ovulation, progestin was introduced to a novel ovarian stimulation regimen for infertility treatment. However, the local action of progestin on the ovary and its effect on clinical outcomes have not been described. Objectives: The influence of progesterone administration on clinical oocyte outcomes and the mechanisms involved in the coordination of progesterone and follicle stimulating hormone (FSH) on follicle growth and oocyte yields were investigated. Methods: Clinical outcomes of patients undergoing ovarian stimulation for in vitro fertilization were analyzed. The murine ovarian stimulation model and follicle culture system were used to evaluate the effects of progesterone on oocyte yield, follicle development, granular cell proliferation, and hormone secretion. Phospho-specific protein microarrays were used to explore involved signaling pathways. Results: Progesterone decreased clinical oocyte yields, and yields were rescued with an increased dose of human menopausal gonadotropin. Administration of progesterone inhibited murine granular cell proliferation and reduced the growth rate of follicles; both of which were rescued by FSH. The phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) were identified as pivotal signaling pathways to integrate progesterone into the FSH signaling network in granular cells. Conclusion: Progesterone inhibited granular cell proliferation and antral follicle growth during ovarian stimulation, and subsequently influenced oocyte outcomes in the clinical setting. Progesterone coordinated with FSH to regulate follicle growth through PI3K/AKT and MAPK signaling pathways. These findings advance our knowledge regarding the ovarian response to gonadotropins during progestin-primed ovarian stimulation and create an opportunity to manipulate individual oocyte yields.

Effects of Early Cumulus Cell Removal on Treatment Outcomes in Patients Undergoing In Vitro Fertilization: A Retrospective Cohort Study.

Background: Early cumulus cell removal combined with early rescue intracytoplasmic sperm injection (ICSI) has been widely practiced in many in vitro fertilization (IVF) centers in China in order to avoid total fertilization failure. However, uncertainty remains whether the pregnancy and neonatal outcomes are associated with early cumulus cell removal. Objectives: To investigate if early cumulus cell removal alone after 4 hours co-incubation of gametes (4 h group), has detrimental effect on the pregnancy and neonatal outcomes in patients undergoing IVF, through a comparison with conventional cumulus cell removal after 20 hours of insemination (20 h group). Methods: This retrospective cohort study included 1784 patients who underwent their first fresh cleavage stage embryo transfer at the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital from June 2016 to December 2018 (4 h group, n=570; 20 h group, n=1214). A logistic regression analysis was performed to examine the independent association between early cumulus cell removal and pregnancy outcomes after adjustment for potential confounders. The neonatal outcomes between the two groups were compared. Results: When compared with the 20 h group, the 4 h group had similar pregnancy outcomes, including rates for biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage, ectopic pregnancy, multiple pregnancy, live birth. There were 1073 infants delivered after embryo transfer (4 h group, n=337; 20 h group, n=736). Outcomes in both groups were similar for both singleton and twin gestations, including preterm birth rate and very preterm birth rate, mean birth weight, mean gestational age, sex ratio at birth and rate of congenital birth defects. In addition, findings pertaining to singleton gestations were also similar in the two groups for Z-scores (gestational age- and sex-adjusted birth weight), rates of small for gestational age, very small for gestational age, large for gestational age and very large for gestational age infants. Conclusions: In this study early cumulus cell removal alone was not associated with adverse pregnancy and neonatal outcomes. From this perspective, early cumulus cell removal to assess for a potential early rescue ICSI is therefore considered to be a safe option in patients undergoing IVF.

Effect of male age on pregnancy and neonatal outcomes in the first frozen-thawed embryo transfer cycles of IVF/ICSI treatment.

BACKGROUND: The effect of male age on pregnancy outcomes after assisted reproductive technology (ART) treatment shown in the previous literature is controversial. In addition, the influence of male age on neonatal outcomes following ART treatment has less been investigated. OBJECTIVES: The aim of this study was to evaluate the effect of male age on reproductive and neonatal outcomes in couples following ART treatment. MATERIALS AND METHODS: A retrospective cohort study was performed in two centers for assisted reproduction from June 2010 to February 2019. A total of 5512 frozen-thawed embryo transfer (FET) cycles were included according to the criteria. The primary outcome measures were pregnancy and neonatal outcomes. Patients were categorized into five groups according to male age (younger than 30, 31-35, 36-40, 41-45, and older than 45), and the group younger than 30 years old was treated as the reference group. RESULTS: The logistic regression analysis showed that clinical pregnancy and live birth were all no statistic difference among the male age-groups compared with the reference group (p values, 0.743, 0.979, 0.948, 0.28; p values, 0.823, 0.342, 0.817, 0.381, respectively). Furthermore, no significant differences were found in the preterm birth rate, child sex, neonatal malformation, birth weight, and gestational age (p > 0.05). The advanced male age was not associated with a higher risk of adverse neonatal outcomes. DISCUSSION AND CONCLUSION: This study showed that there were no effects of male age on pregnancy or neonatal outcomes in infertile couples following their first FET cycles when females were younger than 36 years old.

Novel mutations in ZP1: Expanding the mutational spectrum associated with empty follicle syndrome in infertile women.

Empty follicle syndrome (EFS) is a serious and complex reproductive complication for infertile women suffering from the recurrent failure of oocyte retrieval in an in vitro fertilization procedure, and its pathogenesis remains obscure. Increasing evidence highlights the genetic basis of EFS occurrence. In this study, we identified two novel missense mutations (c.1127G > A, p.C376Y and c.325C > T, p.R109C), two novel frameshift mutations (c.800_801delAG, p.E267Gfs*80 and c.1815_1825delGGTCCTTTTGC, p.V606Afs*42), one novel nonsense mutation (c.199G > T, p.E67Ter), and three reported mutations (c.769C > T, p.Q257Ter; c.1430 + 1G > T, p.C478Ter and c.1169_1176delTTTTCCCA, p.I390Tfs*16) in five unrelated probands, showing similar EFS manifestations, which expands the mutational spectrum of individuals with autosomal recessive ZP1. Current research will provide a better understanding of the biological functions of ZP1, and some insight into the determination of ZP1 variation as an additional rule for assessing the EFS disease.

Pregnancy and neonatal outcomes of morphologically grade CC blastocysts: are they of clinical value?

PURPOSE: To study if the transfer of morphological grade-CC blastocyst is effective and safe. METHODS: This retrospective study included 2585 frozen-thawed embryo transfer (FET) cycles with grade-BB blastocysts and 102 FET cycles with grade-CC blastocysts during the period from January 2006 to December 2017. Pregnancy and neonatal outcomes of couples in two groups were analyzed before and after propensity score matching. RESULTS: Pregnancy outcomes showed no significant difference in the rates of biochemical pregnancy, ectopic pregnancy, miscarriage, multiple gestation, gestational age (P > 0.05). However, the rates of intrauterine implantation, clinical pregnancy, and live birth were significantly lower in the grade-CC blastocyst transfer group than those in the grade-BB blastocyst transfer group (18.9% vs 46.0%, 21.6% vs 51.3%, 16.7% vs 41.4%, all P < 0.001, respectively) before and after propensity score matching. The assessment of neonatal outcomes showed no statistically significant differences in the birth weight, low birth weight, early-neonatal death, and birth defect, etc., similar results were also observed in the two matched cohorts. CONCLUSION: Morphologically grade-CC blastocysts should be transferred rather than discarded, resulting in acceptable pregnancy and neonatal outcomes, which is beneficial to infertile patients suffering from repeated poor-quality embryos.

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.

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.

The live birth and neonatal outcomes in the subsequent pregnancy among patients with adverse pregnancy outcomes in first frozen embryo transfer cycles.

PURPOSE: To explore whether the adverse pregnancy outcomes in first frozen embryo transfer (FET) cycles affect live birth and neonatal outcomes in the subsequent pregnancy? METHODS: This was a retrospective study. Women with a history of adverse pregnancy outcomes in first FET cycles started their subsequent embryo transfer cycles. The adverse pregnancy outcomes included biochemical pregnancy, ectopic pregnancy, and first-trimester pregnancy loss. The main outcomes of present study were live birth rate and neonatal outcomes. RESULTS: Results showed patients with first-trimester pregnancy loss in first FET cycles had a 95 percent greater chance of live birth in subsequent FET cycles (OR 1.95, 95% CI 1.33-2.88). However, the biochemical pregnancy/ectopic pregnancy in initial FET cycles did not affect the chance of live birth in second cycles (biochemical pregnancy: OR 1.21, 95% CI 0.82-1.77; ectopic pregnancy: OR 1.06, 95% CI 0.55-2.05). The neonatal outcomes of singletons were not affected by the number of embryo transfer cycles. CONCLUSIONS: Patients with first-trimester pregnancy loss in first FET cycle had a greater chance of live birth in second FET cycles, but the biochemical pregnancy/ectopic pregnancy in first FET cycles did not significantly affect the live birth in second FET cycles. The three types of adverse pregnancy outcomes in first FET cycles did not affect neonatal outcomes in the second cycles.

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.

Effect of Maternal Advanced Endometriosis on Risk of Congenital Malformations for Infants Born After in vitro Fertilization and Frozen-Thawed Embryo Transfer: Analysis of 28,600 Newborns.

Background: Endometriosis is one of the most challenging diseases for doctors helping infertile women conceive, which has become a common method to help maternal endometriosis-associated infertility. Women with advanced endometriosis possess a higher risk of several adverse outcomes both during pregnancy and at the time of delivery. Whether endometriosis gives rise to a higher occurrence of congenital abnormalities in infants via in vitro fertilization and frozen-thawed embryo transfer (IVF-ET) remains unknown. Methods: Data collected on 22,865 women undergoing IVF using a freeze-all strategy from 2007 to 2017 were analyzed to estimate the rate of congenital malformations. We used an adjusted OR to compare the fertility outcomes of women with advanced endometriosis to the control group. Results: We studied 1,495 infants born from women with advanced endometriosis and 27,105 infants born from endometriosis-free women. There was a 1.557-fold risk that the infants with advanced maternal endometriosis would develop a congenital malformation (adjusted OR: 1.557, 95% CI: 1.03-2.35). Compared with singletons, twins were 1.957 times more likely to experience an adverse outcome (OR: 1.957, 95% CI: 1.561-2.455). When analyzing specific categories of birth defects, the proportion of circulatory system defects was higher than the other categories of birth defects in total (0.56%), followed by musculoskeletal system defects (0.15%). Conclusions: Maternal advanced endometriosis might increase the risk of congenital malformations for infants born after IVF-ET. The organ system most frequently affected by congenital malformations was the cardiovascular system, followed by the musculoskeletal system.

Pregnancy and neonatal outcomes of artificial oocyte activation in patients undergoing frozen-thawed embryo transfer: a 6-year population-based retrospective study.

PURPOSE: To evaluate the impact of artificial oocyte activation (AOA) in pregnancy and neonatal outcomes in infertile patients undergoing cryopreserved embryo transfer. METHOD: This retrospective study included 5686 patients' transferred embryos from routine intracytoplasmic sperm injection (ICSI) and 194 patients' transferred embryos from ICSI combined with AOA (ICSI-AOA) from January 2011 to December 2016. Pregnancy and neonatal outcomes of couples undergoing routine ICSI or ICSI-AOA were analyzed before and after propensity score matching. Artificial oocyte activation was performed with ionomycin. RESULTS: The pregnancy outcomes showed no significant difference in the rates of biochemical pregnancy, clinical pregnancy, implantation, miscarriage, ectopic pregnancy, multiple pregnancy, and live births between the routine ICSI and ICSI-AOA groups before and after propensity score matching, respectively. The assessment of neonatal outcomes showed no statistically significant differences in the birth defect rate, birth weight, gestational age, preterm birth rate, early-neonatal death rate, and fetal sex ratio between the two groups, and similar results were also observed in the two matched cohorts. CONCLUSION: Artificial oocyte activation with ionomycin does not adversely affect pregnancy and neonatal outcomes in patients undergoing frozen-thawed embryo transfer, which is beneficial to clinicians counseling patients on the risks of artificial oocyte activation.