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Purpose: To explore the pathogenesis of oocyte maturation defects., Methods: Whole exome sequencing was conducted to identify potential variants, which were then confirmed within the pedigree through Sanger sequencing. The functional characterization of the identified variants responsible for the disease, including their subcellular localization, protein levels, and interactions with other proteins, was verified through transient transfection in HeLa cells in vitro. Additionally, we employed real-time RT-PCR and single-cell RNA sequencing to examine the impact of ZFP36L2 pathogenic variants on mRNA metabolism in both HeLa cells and mouse or human oocytes., Results: A novel compound heterozygous variant in ZFP36L2 (c.186T > G, p.His62Gln and c.869 C > T, p.Pro290Leu) was discovered in a patient with oocyte maturation defects. Our findings indicate that these variants lead to compromised binding capacity of the ZFP36L2-CONT6L complex and impaired mRNA degradation in HeLa cells and mouse oocytes. Furthermore, we characterized the changes in the human oocyte transcriptome associated with ZFP36L2 variants, with a particular emphasis on cell division, mitochondrial function, and ribosome metabolism., Conclusions: This study broadens the mutation spectrum of ZFP36L2 and constitutes the first report of human oocyte transcriptome alterations linked to ZFP36L2 variants. In conjunction with existing knowledge of ZFP36L2, our research lays the groundwork for genetic counseling aimed at addressing female infertility., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Study Objective: To investigate whether different timings of GnRH-a downregulation affected assisted reproductive outcomes in infertile women with moderate-to-severe intrauterine adhesions (IUAs) accompanied by adenomyosis., Design: A retrospective case series., Setting: An assisted reproductive technology center., Patients: The study reviewed 123 infertile women with moderate-to-severe IUAs accompanied by adenomyosis undergoing their first frozen-thawed embryo transfer (FET) cycles between January 2019 and December 2021., Measurements and Main Results: The majority of patients had moderate IUA (n=116, 94.31%). The average Basal uterine volume was 73.58 ± 36.50 cm 3 . The mean interval from operation to the first downregulation was 21.07 ± 18.02 days (range, 1-79 days). The mean duration of hormone replacement therapy (HRT) was 16.93 ± 6.29 days. The average endometrial thickness on the day before transfer was 10.83 ± 1.75 mm. A total of 70 women achieved clinical pregnancy (56.91%). Perinatal outcomes included live birth (n=47, 67.14%), early miscarriage (n=18, 25.71%), and late miscarriage (n=5, 7.14%). The time interval between uterine operation and the first downregulation was not a significant variable affecting live birth. Maternal age was the only risk factor associated with live birth (OR:0.89; 95% CI: 0.79-0.99, P=0.041)., Conclusions: The earlier initiation of GnRH-a to suppress adenomyosis prior to endometrial preparation for frozen embryo transfer did not negatively impact repair of the endometrium after resection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Peng, Chen, Wang, Dong, Liu, Yao, Zhang, Li, Cai, Li, Lin and Gong.)

Fertilization of the egg by the sperm is the first vital stage of embryogenesis. In mammals, only one sperm is incorporated into the oocyte. Polyspermy is a key anomaly of fertilization that is generally lethal to the embryo. To date, only a few causative genes for polyspermy have been reported. In a recent study, a homozygous variant in astacin-like metalloendopeptidase (ASTL), which encodes the ovastacin enzyme that cleaves ZP2 to prevent polyspermy, was found to be associated with female infertility characterized by polyspermy in vitro. Herein, we identified two ASTL variants in a Chinese woman likely responsible for her primary infertility and polyspermy in in vitro fertilization. Both variants were located within the key catalytic domain and predicted to alter hydrogen bonds, potentially impairing protein stability. Moreover, expression and immunoblot analyses in CHO-K1 cells indicated abnormal ovastacin zymogen activation or decreased enzyme stability. Intracytoplasmic sperm injection treatment successfully bypassed the defect in polyspermy blocking and resulted in a live birth. Our study associates ASTL variants with human infertility and further supports the contribution of this gene to blocking polyspermy in humans. Our findings expand the spectrum of ASTL mutations and should facilitate the diagnosis of oocyte-borne polyspermy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: To obtain quantitative and comprehensive results of the changes in comprehensive ER indicators from ovulation day to transplantation day by ultrasonography during the natural frozen-thawed embryo transfer cycle (FET)., Methods: This is a prospective analysis of 230 infertile women undergoing their first FET cycles from April 2019 to July 2021. To evaluate ER, ultrasound scans were performed on the days of ovulation and embryo transfer for all included patients. All included patients were divided into a pregnancy group and a nonpregnancy group according to whether clinical pregnancy was achieved. The ER changes from ovulation day to transplantation day in the overall study population (n=230), pregnancy group (n=158) and nonpregnancy group (n=72) were analyzed., Results: In the overall population, type C was predominant on ovulation day, but type B was the most common on transplantation day (P<0.001). From ovulation day to transplantation day, endometrial thickness was significantly increased (11.26 ± 2.14 vs. 11.89 ± 2.08 mm, P<0.001), but endometrial volume (4.26 ± 1.75 vs. 4.03 ± 1.62 ml, P<0.001), endometrial VI (1.34 ± 1.64 vs. 0.95 ± 1.99, P<0.001), VFI (0.47 ± 0.72 vs. 0.40 ± 1.03, P<0.001), subendometrial VI (5.04 ± 3.89 vs. 3.29 ± 2.92, P<0.001), FI (34.07 ± 4.61 vs. 33.41 ± 5.30, p=0.004), VFI (2.07 ± 2.65 vs. 1.19 ± 1.19, P<0.001) and frequency of endometrial peristalsis (2.90 ± 1.44 vs. 1.40 ± 1.41, P<0.001) were significantly decreased. In the pregnancy group, the changes in all ultrasound parameters were in the same direction as those in the overall population. In the nonpregnancy group, except for endometrial volume and VI, which showed no difference, other ultrasound parameters showed the same direction of change as those in the overall population. No significant difference was found in the pregnancy probability among the different absolute change groups., Conclusion: During a natural cycle, the morphology of the endometrium changes mostly from type C to type B, the endometrial thickness increases, and the volume decreases. The blood supply of the endometrium, the subendometrial 5 mm and the frequency of peristalsis decrease from ovulation day to transplantation day. Compared with the nonpregnancy group, the pregnancy group tended to have more obvious decreases in endometrial volume and blood flow perfusion. However, these endometrial changes do not mean that pregnancy is bound to occur. endometrial receptivity, in vitro fertilization, frozen-thawed embryo transfer, natural cycle, ultrasound evaluation, ovulation day, transplantation day., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Peng, Mao, Li, Gong and Ouyang.)

Background: Detailed knowledge of the changes in endometrial immune cells during the window of implantation in unexplained recurrent implantation failure (RIF) patients, the functions performed by immune cells, and the interactions between them is largely lacking. This study aimed to classify RIF patients and explore the mechanism through endometrial immune profiling and RNA-seq analysis., Methods: This study enrolled a total of 172 patients, comprising 144 women with unexplained RIF and 28 fertile women. Endometrial samples were collected using endometrial scratching at the mid-luteal phase before in vitro fertilization treatment or pregnancy. Transcriptome sequencing and immunohistochemical staining of endometrial immune cells including natural killer (NK) cells, macrophages, T cells, and B cells were performed., Main Outcome Measure(s): Comparison of the percentage of endometrial immune cells and the RNA-seq information between RIF patients and fertile control patients., Result(s): The proportions of uterine CD56 + uNK cells, CD57 + NKT cells, CD68 + macrophages, and CD19 + B cells were significantly elevated in RIF patients. In addition, the number of positive CD68 glandular lumens was significantly higher in RIF patients than in the fertile group. In addition, based on this result, we classified RIF patients into three categories., Conclusion(s): Hyperactivation of endometrial immune cells may be associated with reduced endometrial tolerance and recurrent implantation failure, affecting pregnancy outcomes in RIF patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Objectives: To investigate the effectiveness of surgery for septate uterus in infertile patients before in vitro fertilization-embryo transfer (IVF-ET)., Methods: The data of 937 infertile patients with septate uterus and achieved singleton pregnancy after IVF-ET from January 2014 to December 2015 were retrospectively analyzed. Thousand five hundred seventy-eight infertile patients with a normal uterus who achieved singleton pregnancy during the same period were selected as the control group. Patients with septate uterus were divided into two groups according to whether the septum was resected. The pregnancy and perinatal outcomes of the surgical group and the nonsurgical group were compared with the control group. The secondary infertility patients who were surgically corrected septa were also chosen as self-controls and an analysis was performed on their fertility outcomes pre- and post-surgery., Results: Compared with the control group, the surgical group had increased rates of early miscarriage, preterm delivery, and low birthweight and a significantly reduced live birth rate (P < .05). The outcomes of the nonsurgical and control groups were similar. Using secondary infertility patients who were surgically corrected septa as self-controls, after surgery, the rates of miscarriage and ectopic pregnancy were significantly lower and the live birth rate was significantly higher (P ≤ .001); however, perinatal mortality was not significantly different before and after surgery., Conclusions: Patients with a septum depth greater than 10 mm or 5-10 mm associated with a history of unexplained recurrent miscarriage, IVF failure, or infertility might benefit from resection of the uterine septum with hysteroscopic metroplasty., (© 2022 The Authors. Journal of Ultrasound in Medicine published by Wiley Periodicals LLC on behalf of American Institute of Ultrasound in Medicine.)

Background: Recurrent preimplantation embryo developmental arrest (RPEA) is the most common cause of assisted reproductive technology treatment failure associated with identified genetic abnormalities. Variants in known maternal genes can only account for 20%-30% of these cases. The underlying genetic causes for the other affected individuals remain unknown., Methods: Whole exome sequencing was performed for 100 independent infertile females that experienced RPEA. Functional characterisations of the identified candidate disease-causative variants were validated by Sanger sequencing, bioinformatics and in vitro functional analyses, and single-cell RNA sequencing of zygotes., Results: Biallelic variants in ZFP36L2 were associated with RPEA and the recurrent variant (p.Ser308&#95;Ser310del) prevented maternal mRNA decay in zygotes and HeLa cells., Conclusion: These findings emphasise the relevance of the relationship between maternal mRNA decay and human preimplantation embryo development and highlight a novel gene potentially responsible for RPEA, which may facilitate genetic diagnoses., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Study Question: What is the genetic basis of female infertility involving abnormal oocyte morphology with the production of a large first polar body (PB1)?, Summary Answer: The homozygous missense variant (c.791C>G) and compound missense variants (c.596A>T and c.875C>T) in MOS proto-oncogene, serine/threonine kinase (MOS) (Online Mendelian Inheritance in Man (OMIM) reference: 190060; NM&#95;005372.1) are responsible for abnormal oocyte morphology with the production of a large PB1 to cause infertility in women., What Is Known Already: MOS, an oocyte-specific gene, encodes a serine/threonine-protein kinase that directly phosphorylates mitogen-activated protein kinase (MAPK) kinase (MEK) to activate MAPK (also called extracellular-signal-regulated kinase (ERK)) signal cascade in the oocyte. Female mice lacking Mos remained viable, but infertile because of oocyte symmetric division, spontaneous parthenogenetic activation and early embryonic arrest. Recently, two independent studies demonstrated that female infertility with early embryonic arrest and fragmentation can be caused by biallelic mutations in MOS. However, so far, MOS variants have not been associated with the phenotype of large PB1 extrusion in human oocytes to contribute to female infertility., Study Design, Size, Duration: Two independent infertile families characterized by the presence of large PB1 in oocytes were recruited between December 2020 and February 2022., Participants/materials, Setting, Methods: Genomic DNA was extracted from the peripheral blood samples of the subjects for whole-exome sequencing. Pedigree analysis was validated by Sanger sequencing. Then, the pathogenic effects of the MOS variants on MOS protein properties and ERK1/2 activation were determined in HEK293 cells and mouse oocytes., Main Results and the Role of Chance: We identified three rare missense variants in MOS, including a homozygous missense variant (c.791C>G) from Patient 1 in Family 1 and two compound missense variants (c.596A>T and c.875C>T) from twin sisters in Family 2. The MOS variants followed a recessive inheritance pattern in infertile patients. All three patients displayed a high percentage of large PB1 extrusion in the oocytes. The three MOS variants could not activate MEK1/2 and ERK1/2 in oocytes and HEK293 cells. In addition, when compared with wild-type MOS, the MOS variants decreased the MOS protein level and attenuated the binding capacity with MEK1. Microinjection of wild-type human MOS complementary RNAs (cRNAs) reversed the symmetric division of oocytes after siMos treatment. In contrast, the three MOS variants demonstrated no rescuing ability., Large Scale Data: N/A., Limitations, Reasons for Caution: Owing to the scarcity of human oocyte samples and the associated ethical restrictions, we could not perform the rescue attempt for the study patients., Wider Implications of the Findings: Our findings expand the genetic and phenotypic spectrum of MOS variants in causing female infertility. Our study findings facilitate the early genetic diagnosis of abnormal oocyte morphology characterized as large PB1 that eventually causes infertility in women., Study Funding/competing Interest(s): This study was supported by the National Natural Science Foundation of China (82071640 and 82001633), Natural Science Foundation of Zhejiang Province (LD22C060001), the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2005), China Postdoctoral Science Foundation (2020M682575 and 2021T140198), the Changsha Municipal Natural Science Foundation (kq2007022) and Hunan Provincial Grant for Innovative Province Construction (2019SK4012). None of the authors declare any competing interests., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is associated with an increased risk of preterm (33rd-37th gestational week) and early preterm birth (20th-32nd gestational week). The underlying general and procedure related risk factors are not well understood so far. 4328 infertile women undergoing IVF/ICSI were entered into this study. The study population was divided into three groups: (a) early preterm birth group (n = 66), (b) preterm birth group (n = 675) and (c) full-term birth group (n = 3653). Odds for preterm birth were calculated by stepwise multivariate logistic regression analysis. We identified seven independent risk factors for preterm birth and four independent risk factors for early preterm birth. Older (> 39) or younger (< 25) maternal age (OR: 1.504, 95% CI 1.108-2.042, P = 0.009; OR: 2.125, 95% CI 1.049-4.304, P = 0.036, respectively), multiple pregnancy (OR: 9.780, 95% CI 8.014-11.935, P < 0.001; OR: 8.588, 95% CI 4.866-15.157, P < 0.001, respectively), placenta previa (OR: 14.954, 95% CI 8.053-27.767, P < 0.001; OR: 16.479, 95% CI 4.381-61.976, P < 0.001, respectively), and embryo reduction (OR: 3.547, 95% CI 1.736-7.249, P = 0.001; OR: 7.145, 95% CI 1.990-25.663, P = 0.003, respectively) were associated with preterm birth and early preterm birth, whereas gestational hypertension (OR: 2.494, 95% CI 1.770-3.514, P < 0.001), elevated triglycerides (OR: 1.120, 95% CI 1.011-1.240, P = 0.030) and shorter activated partial thromboplastin time (OR: 0.967, 95% CI 0.949-0.985, P < 0.001) were associated only with preterm birth. In conclusion, preterm and early preterm birth risk factors in patients undergoing assisted IVF/ICSI are in general similar to those in natural pregnancy. The lack of some associations in the early preterm group was most likely due to the lower number of early preterm birth cases. Only embryo reduction represents an IVF/ICSI specific risk factor., (© 2022. The Author(s).)

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F-actin assembly. Using oocyte-specific Erk1/2 knockout mice, we verified that MOS-ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS-ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Many factors impede embryonic implantation, and excluding obvious known factors such as chronic endometritis, the immune status of the endometrium may be related to pregnancy. Although an abundantly large number of immune cells infiltrate the endometrium during the secretory phase, whether these immune cells can be used as a predictor of prognosis in ART has not yet been clarified. In the present study we therefore retrospectively analyzed 97 CD138-negative women with a previous fresh-embryo-transfer failure. We assessed the expression of CD56+ uNK cells, CD16+ NK cells, CD57+ NK cells, CD68+ pan-macrophages, CD163+ M2 macrophages, CD4+T cells, CD8+T cells, FOXP3+ regulatory T cells, and CD19+ B cells in the endometrium by IHC to evaluate mid-luteal endometrial immune cells as prognostic indicators of pregnancy outcome in the next frozen-embryo-transfer cycle. CD19-positive cells and the intraglandular CD163-positivity rate increased significantly in the clinically non-pregnant group (0.47 % vs. 0.20 %, P =  0.021; 61 % vs. 30 %, P =  0.017). The ratios of CD4/CD8 were also higher in the non-pregnant group (1.96 vs. 1.45, P =  0.005).The area under the ROC curve of CD19 cell number alone, the intraglandular CD163-positivity alone, and CD19 number combined with the intraglandular CD163-positivity were 0.692 (95 % CI, 0.55-0.834), 0.661 (95 % CI, 0.514-0.809), and 0.748 (95 % CI, 0.614-0.882), respectively. The optimal cut-off value of CD19 was 0.464 %, and the clinical pregnancy rate and live-birth rate diminished significantly when the CD19 level was above this cut-off value. Our study suggests that CD19-positive cells and intraglandular CD163-positivity can be used as prognostic indicators of pregnancy outcome in CD138-negative patients who experienced first-fresh-embryo transfer failure., (Copyright © 2021. Published by Elsevier B.V.)

Purpose: TUBB8 is a gene that is frequently analysed in the genetic diagnosis of female infertility; 102 variants of this gene have been identified. However, the evaluation of its pathogenicity and the resulting phenotypes vary. Here, we aimed to identify novel TUBB8 variants as well as to summarize the reported variants and phenotypes in order for them to be included in genetic counselling analyses., Methods: We performed whole exome sequencing to screen for candidate variants in 100 infertile female subjects and 100 controls who were able to conceive naturally. All variants were confirmed by Sanger sequencing. The effects of the variants in oocytes/arrested embryos were assessed by morphological observations, polar body biopsies, and chromosome analysis. A molecular modelling analysis was used to evaluate the possible effects of variants on protein secondary structure., Results: We identified 29 TUBB8 variants, of which 20 were novel and five were maternally inherited. We identified three of a total of six recurrent variants that were specific for complete cleavage failure. Moreover, we obtained evidence that TUBB8 variants with large polar bodies had chromosome segregation errors., Conclusions: Our study expands the spectrum of TUBB8 variants, particularly for embryonic arrest. Together with the extant knowledge of TUBB8 variants, this study provides a foundation for the genetic counselling of female infertility., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The genetic causes of the majority of cases of female infertility caused by premature ovarian insufficiency (POI) are unknown., Objective: To identify the genetic causes of POI in 110 patients., Methods: Whole-exome sequencing was performed on 110 patients with POI, and putative disease-causative variants were validated by Sanger sequencing. Bioinformatic and in vitro functional analyses were performed for functional characterisation of the identified candidate disease-causative variants., Results: We identified two homozygous variants (NM&#95;001040274: c.150&#95;151del (p.Ser52Profs*7), c.999A>G (p.Ile333Met)) in SYCP2L in two patients, which had co-segregated with POI in these families. Bioinformatic analysis predicted that the two variants are deleterious, and in vitro functional analysis showed that mutant SYCP2L proteins exhibited mislocalisation and loss of function., Conclusions: SYCP2L is a novel gene found to be responsible for human POI. Our findings provide a potential molecular marker for POI and improve the understanding of the genetic basis of female infertility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To measure free and total 25-hydroxyvitamin D [25(OH)D] immediately before embryo transfer and analyze its association with early pregnancy outcome parameters such as biochemical pregnancy, implantation rate, and clinical pregnancy rates in women undergoing fresh embryo transfer after their first ovarian hyperstimulation., Design: Prospective cohort study., Setting: Academically affiliated private fertility center., Patient(s): A total of 2,569 women undergoing fresh embryo transfer after ovarian hyperstimulation., Interventions(s): None., Main Outcome Measure(s): The study end points were biochemical pregnancy rate, implantation rate, clinical pregnancy rate, ectopic pregnancy rate, early miscarriages, and ongoing pregnancy rate. Free and total 25(OH)D concentrations were measured 1 day before embryo transfer., Result(s): Total 25(OH)D correlated with free 25(OH)D. Total and free 25(OH)D serum concentrations were similar in those patients reaching and not reaching the study outcomes (biochemical pregnancy rate, implantation rate, clinical pregnancy rate, ectopic pregnancy rate, early miscarriages, and ongoing pregnancy rate). There was likewise no statistical difference when analyzing the frequency of all study outcomes in quintiles of either total or free 25(OH)D. In addition, the study population was divided into three groups according to the total vitamin D status based on clinical practice guideline. All outcomes were similar in women with adequate, insufficient, and deficient total 25(OH)D. Multiple linear regression analysis considering confounding likewise indicated no association of free or total vitamin D with any of the study outcomes., Conclusion(s): Neither free nor total 25(OH)D concentration at embryo transfer was associated with successful embryo implantation in women undergoing fresh transfer after ovarian hyperstimulation., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The subcortical maternal complex (SCMC) is an oocyte-to-embryo-specific maternal functional module. Some variants of SCMC genes that contribute to preimplantation embryonic arrest have been identified. However, more novel variants should be identified to broaden the genetic and phenotypic spectrum of SCMC genes and establish their roles in embryonic development. We identified 13 novel variants in the SCMC genes, TLE6, NLRP5, NLRP2, and PADI6, from 10 of a total of 50 infertile females with recurrent preimplantation embryonic arrest. Six variants in TLE6 were found in five patients with embryonic arrest, accompanied by direct cleavage and severe fragmentation at the cleavage stage. Three patients carried NLRP5 variants, and one patient each who carried NLRP2 and PADI6 variants had subsequent poor or failed fertilization and cleavage arrest with a relatively lower ratio of severely fragmented embryos. Our findings expand the genetic and phenotypic spectrum of SCMC genes associated with human embryogenesis and might help lay the foundation for the genetic diagnosis of female infertility., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM&#95;017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475&#95;478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the pathogenesis of the recurrent preimplantation embryonic arrest characterized by direct cleavage., Methods: Two affected individuals underwent time-lapse imaging to observe the cleavage behaviors in their final ICSI attempts. In addition, both patients were subjected to whole-exome sequencing. After the identification of possible causative genes, molecular modeling analyses were used to evaluate the possible effects of candidate mutations on protein secondary structure., Results: All the bipronucleated (2PN) zygotes from both individuals presented multiple abnormal cleavage behaviors, particularly direct cleavage (DC) and subsequent cleavage arrest. Mutation analysis identified one new frameshift mutation c.1521dupC (p.S508Qfs*5) and two missense mutations c.A1117C and c.C1708T (p.T373P and p.R570C, respectively) of the PADI6 gene, which were in the protein-arginine deiminase (PAD) domain and highly conserved., Conclusion: This study expands the mutation spectrum of PADI6 and is the first to propose that the preimplantation embryonic arrest with concomitant abnormal cleavage behaviors, especially DC, maybe associated with PADI6 mutations.

Objective: To investigate the effectiveness of Tornado and Hilal (Cook Medical, Bloomington, IN) hysteroscopic proximal tubal occlusion (HPTO) for hydrosalpinx (HX) treatment before in vitro fertilization (IVF) and embryo transfer (ET) in patients with dense pelvic adhesions or low ovarian reserve., Design: A retrospective study of patients treated between May 2014 and May 2016 (Canadian Task Force classification III)., Setting: A university-affiliated center., Patients: One hundred fifty women with unilateral or bilateral HX who were not candidates for laparoscopic surgery., Intervention: Tornado or Hilal HPTO before IVF-ET and/or frozen ET., Measurements and Main Results: The main outcome was the cumulative live birth rate. Tornado or Hilal placement was successful for 143 (95.3%) patients. Of the 132 patients who underwent 204 ET cycles, 86 (65.2%) conceived, and 78 (59.1%) resulted in live births. The overall early and late miscarriage rates per clinical pregnancy were 12.8% and 1.1%, respectively. The ectopic pregnancy rate was 2.3%. No major complications occurred., Conclusion: Tornado or Hilal HPTO is an effective alternative for treating HX before IVF-ET/frozen ET in patients with dense pelvic adhesions or low ovarian reserve., (Copyright © 2018 AAGL. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the genetic cause of fertilization failure or poor fertilization., Design: Genetic analysis., Setting: University-affiliated center., Patient(s): Twenty-four Chinese women who underwent assisted reproductive technology (ART) and had repeated fertilization failure or poor fertilization., Intervention(s): None., Main Outcome Measure(s): Twenty-four affected patients were subjected to whole-exome sequencing and candidate mutations were validated by Sanger sequencing. Single-cell reverse transcription was used to analyze the functional characterization of the splice-site mutation in vivo. Evolutionary conservation and molecular modeling analyses were used to predict the impact of missense mutations on secondary protein structure. Immunofluorescence was used to analyze the protein levels of WEE2 and phosphorylated CDC2., Result(s): Biallelic mutations in WEE2 were identified in 5 of 24 (20.8%) Chinese patients with fertilization failure or poor fertilization. Among these individuals we found a novel splice-site mutation, two novel missense mutations, and a previously reported frame-shift mutation. Splicing mutation c.1136-2A>G of WEE2 caused an alteration of the reading frame and introduced a premature stop codon (p.Gly379Glufs*6/p.Asp380Leufs*39). The missense mutations c.585G>C (p.Lys195Asn) and c.1228C>T (p.Arg410Trp) produced obvious changes in secondary protein structures. Immunostaining indicated that mutated WEE2 resulted in the loss of phosphorylated CDC2. The phenotypes of women carrying WEE2 mutations exhibited slight variability, from total fertilization failure to poor fertilization., Conclusion(s): Novel mutations in the known causative gene WEE2 were identified in 5 of 24 women with fertilization failure or poor fertilization, indicating a high prevalence of WEE2 mutations in Chinese women experiencing fertilization failure or poor fertilization., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: The oocyte-borne genetic causes leading to fertilization failure are largely unknown. We aimed to identify novel human pathogenic variants (PV) and genes causing fertilization failure., Methods: We performed exome sequencing for a consanguineous family with a recessive inheritance pattern of female infertility characterized by oocytes with a thin zona pellucida (ZP) and fertilization failure in routine in vitro fertilization. Subsequent PV screening of ZP2 was performed in additional eight unrelated infertile women whose oocytes exhibited abnormal ZP and similar fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type and mutant proteins were carried out in CHO-K1 cells., Results: Two homozygous s PV (c.1695-2A>G, and c.1691&#95;1694dup (p.C566Wfs*5), respectively) of ZP2 were identified in the affected women from two unrelated consanguineous families. All oocytes carrying PV were surrounded by a thin ZP that was defective for sperm-binding. Immunostaining indicated a lack of ZP2 protein in the thin ZP. Studies in CHO cells showed that both PV resulted in a truncated ZP2 protein, which might be intracellularly sequestered and prematurely interacted with other ZP proteins., Conclusion: We identified loss-of-function PV of ZP2 causing a structurally abnormal and dysfunctional ZP, resulting in fertilization failure and female infertility.

Objective: It has been suggested that a progesterone/estradiol ratio (P/E2) ≥ 1.0 on the day of human chorionic gonadotropin (hCG) administration indicates premature luteinization and might be associated with an adverse pregnancy; however, a lower limit of this ratio has not been determined. We aimed to identify a lower limit of P/E2 that correlates significantly with an increase in adverse pregnancies in patients undergoing a prolonged in vitro fertilization/intracytoplasmic sperm injection therapy., Materials and Methods: This retrospective analysis involved 7451 patients who received the first cycle of in vitro fertilization/intracytoplasmic sperm injection therapy treatment at the Reproductive and Genetic Hospital of Citic-Xiangya between January 2008 and April 2012. Patients were stratified into six groups according to their P/E2 on the day of hCG administration. Primary pregnancy outcomes, rates of implantation, clinical pregnancy, ongoing pregnancies, spontaneous abortions, and live births were recorded. The association between P/E2 on the day of hCG administration and primary pregnancy outcomes was assessed using logistic regression analysis., Results: The rates of implantation (23.85-33.44%), clinical pregnancy (47.42-67.12%), ongoing pregnancy (40.83-61.48%), and live birth (34.40-57.65%) were significantly decreased in patients with a P/E2 < 0.25. These indicators were significantly associated with P/E2, but no significant correlation was observed between P/E2 and early spontaneous abortion rate., Conclusion: P/E2 < 0.25 on the day of hCG administration was associated with adverse pregnancy outcomes in extended treatments of gonadotropin-releasing hormone agonist IVF/ICSI., (Copyright © 2017 Taiwan Association of Obstetrics & Gynecology. Published by Elsevier B.V. All rights reserved.)

This study explored the correlation between serum anti-Müllerian hormone (AMH) concentration and the number of retrieved oocytes after ovarian stimulation in Chinese infertile women undergoing assisted reproductive technology treatment and AMH cut-off values predicting low and high ovarian response. This was a prospective, multi-centre, observational study. A total of 615 subjects were included in nine assisted reproductive centres in China for outcome analysis. Subjects received assisted reproductive technology treatment and used recombinant human FSH (r-HFSH) or r-HFSH plus recombinant LH (rLH) for ovarian stimulation according to conventional treatment regimens. The main outcome variables were correlations between AMH and the number of retrieved oocytes and the cut-off values of AMH predicting low and high ovarian response. Serum AMH concentration was positively correlated with the number of oocytes retrieved in Chinese infertile women treated with IVF/intracytoplasmic sperm injection (ICSI) (Pearson correlation coefficient = 0.4754, P < 0.0001). The optimal AMH cut-off value was 2.6 ng/ml (sensitivity: 81.28%, specificity: 59.51%) in predicting high and normal response, and 1.1 ng/ml (sensitivity: 52.27%, specificity: 87.23%) in predicting low and normal response. In conclusion, serum AMH concentration can be used as a biomarker to predict ovarian response in Chinese infertile women treated with assisted reproductive technology., (Copyright © 2016. Published by Elsevier Ltd.)

Objective: To investigate a forecasting method developed to predict first trimester pregnancy outcomes using the first routine ultrasound scan for early pregnancy on days 27-29 after ET and to determine whether to perform a repeated scan several days later based on this forecasting method., Design: Prospective analysis., Setting: Infertile patients at an assisted reproductive technology center., Patient(s): A total of 9,963 patients with an early singleton pregnancy after in vitro fertilization (IVF)-ET., Intervention(s): None., Main Outcome Measure(s): Ongoing pregnancy >12 weeks of gestation., Result(s): The classification score of ongoing pregnancy was equal to (1.57 × Maternal age) + (1.01 × Mean sac diameter) + (-0.19 × Crown-rump length) + 25.15 (if cardiac activity is present) + 1.30 (if intrauterine hematomas are present) - 47.35. The classification score of early pregnancy loss was equal to (1.66 × Maternal age) + (0.84 × Mean sac diameter) + (-0.38 × Crown-rump length) + 8.69 (if cardiac activity is present) + 1.60 (if intrauterine hematomas are present) - 34.77. In verification samples, 94.44% of cases were correctly classified using these forecasting models., Conclusion(s): The discriminant forecasting models are accurate in predicting first trimester pregnancy outcomes based on the first scan for early pregnancy after ET. When the predictive result is ongoing pregnancy, a second scan can be postponed until 11-14 weeks if no symptoms of abdominal pain or vaginal bleeding are present. When the predictive results suggest early pregnancy loss, repeated scans are imperative to avoid a misdiagnosis before evacuating the uterus., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

This study assessed first the impact of endometrial Tiam1/Rac1 signals and microRNA-22 (miR-22) on embryo implantation in mice, and then the expression of the above three genes in the endometrium during the embryo implantation window in the natural menstrual cycle in women with repeated implantation failure (RIF) after in vitro fertilization treatment. Four hundred fifty-two Kun-ming female mice and 200 women (70 infertility patients with RIF, 130 women as controls) were entered into this study. Endometrial Tiam1/Rac1 signals and miR-22 expression were studied in clinical and mouse samples and serum estrogen (E2) and progesterone (P) were analyzed in clinical subjects. A pregnant mouse model based on an endometrial miR-22 and Tiam1 mRNA expression trend of patients with RIF was constructed and then the embryo implantation numbers were analyzed, and an ovariectomized mouse model was used to assess correlations of expression of these three genes with E2 and P. The results showed that during the embryo implantation window in the natural menstrual cycle, endometrial miR-22 was significantly higher whereas Tiam1/Rac1 signals were notably lower in patients with RIF than in controls, and the P:E2 ratio was statistically lower in the RIF group. Tiam1/Rac1 signal down-regulation and miR-22 up-regulation contributed to the inhibition of embryo implantation in mice. We also found a suppressive effect of miR-22 up-regulation on Tiam1/Rac1 signal expression, and reciprocal regulation of E2 and P for these three genes' expression in mice. In conclusion, miR-22 up-regulation and Tiam1/Rac1 signal down-regulation inhibited embryo implantation in mice; this mechanism may be partially due to the suppressive effect of miR-22 on Tiam1 expression, and is regulated to some extent by serum E2 and P. Our findings provide evidence that endometrial Tiam1/Rac1 signal down-regulation along with miR-22 up-regulation during embryo implantation window in the natural menstrual cycle may be one of the reasons for the failure of embryo implantation in patients with RIF., (© 2015 by the Society for the Study of Reproduction, Inc.)

In an attempt to evaluate the effectiveness of a novel modified ultra-long agonist (ULA) protocol on polycystic ovary syndrome (PCOS) patients undergoing in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI), a retrospective study of 499 women employed with either ULA or conventional long agonist (LA) protocol was analyzed. In high BMI group (>25 kg/m²), the ULA protocol yielded significant higher clinic pregnancy rate (PR) (70.2% versus 50.8%, p < 0.05), implantation rate (52.7% versus 35.7%, p < 0.05) and live birth rate (63.8% versus 39.0%, p < 0.05) when compared with LA protocol. In low BMI group (≤25 kg/m²), the ULA protocol also demonstrated a higher clinic PR (70.8% versus 59.5%, p < 0.05) whereas implantation rate and live birth rate are comparable. Within ULA protocol, the clinic PR, implantation rate and live birth rate are similar between high and low BMI patients. Similarly, the clinic PR and live birth rate demonstrated no significant difference within LA group but there is a significant lower implantation rate (35.7% versus 63.9%, p < 0.05) observed in high BMI patients. No difference in miscarriage rate and severe OHSS rate was found among all groups. In conclusion, ULA protocol benefits the IVF outcomes of PCOS patients with high BMI status.

Objective: To investigate the outcome of in vitro fertilization and embryo transfer (IVF-ET) in special infertile patients following modified super-long down-regulation protocol combined with human menopausal gonadotropin (HMG) stimulation., Methods: Ninety-nine special patients (42 with endometriosis, 35 with PCOS, and 22 with insufficient down-regulation) who underwent modified super-long down-regulation protocol in 2008 were retrospectively analyzed. Gonadotropin releasing hormone analogues (GnRHa, 1.5 mg) was injected intramuscularly in mid-luteal phase twice and HMG was started 25 days later after the second GnRHa injection. Conventional IVF-ET was performed as routine procedure. The clinical outcomes were compared with those of 122 similar patients in the same period., Results: After modified super-long down-regulation, (1) in endometriosis patients, the average gonadotropin (Gn) used was (32.33 +/- 15.11) ampoules, duration of medication was (10.57 +/- 1.88) days, the progesterone (P) level on hCG day was (0.78 +/- 0.44) microg/L, and the clinical pregnancy rate (CPR) was 73.8%; (2) in PCOS patients, the average Gn used was (28.57 +/- 12.07) ampoules, the duration of medication was (11.71 +/- 2.07) days, the P level at hCG day was (0.65 +/- 0.39) microg/L, and the clinical pregnancy rate was 65.7%; (3) in insufficient down-regulation patients, the average Gn used was (26.22 +/- 12.07) ampoules, the duration of medication was (10.01 +/- 1.77) days, the P level at hCG day was (0.71 +/- 0.50) microg/L, and the clinical pregnancy rate was 72.7%. Compared with patients using regular down-regulation protocol, the clinical pregnancy rate was improved significantly and the cost decreased obviously. The clinical pregnancy rate significantly improved compared with that of routine long down-regulation groups., Conclusion: Revised super-long protocol plus HMG is a cost-effective controlled ovary hyperstimulation (COH) regimen for infertile patients with endometriosis, PCOS and insufficient down-regulation.

Objective: To investigate the occurrence of ectopic pregnancy among women who received in vitro fertilization and assess the influential factors., Methods: The indications, methods of assisted conception and ectopic types were analyzed retrospectively after the patients received in vitro fertilization and embryo transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), or freezing-thawing embryo transfer (FET)., Results: A total of 6007 embryo transfers were performed, and 2322 (38.7%) clinical pregnancies were obtained. Ninety-four (4.05%) of them were ectopic pregnancies; and 92 were tubal pregnancies. The occurrence rate was 3.96%, which constituted 97.87% (92/94) of all ectopic pregnancies. There were 2 cases of other parts: one in abdominal cavity and the other in cornual pregnancy with the occurrence rate of 0.86%, constituting 2.32% (2/94). Twenty heterotopic pregnancies occurred (0.86%), constituting 21.28% (20/94). Among all ectopic pregnancies, the assisted conception of 86 cases was tubal pathology and/or pelvic adherence (91.49%), and 24 patients had a history of ectopic pregnancy (25.53%). The differences of clinical pregnancy rates between IVF-ET, ICSI and FET were not significant (P>0.05). The ectopic rate of IVF-ET group was significantly higher than that of ICSI or FET group (P<0.05), respectively. The ectopic rate in FET group was also higher than that in ICSI group (P<0.05)., Conclusion: The occurrence rate of ectopic pregnancy after IVF is higher than that of spontaneous pregnancy, and the main cause for ectopic pregnancy is the tubal pathological changes.