Variants in NLRP2 and ZFP36L2, non-core components of the human subcortical maternal complex, cause female infertility with embryonic development arrest.

The subcortical maternal complex (SCMC), which is vital in oocyte maturation and embryogenesis, consists of core proteins (NLRP5, TLE6, OOEP), non-core proteins (PADI6, KHDC3L, NLRP2, NLRP7), and other unknown proteins that are encoded by maternal effect genes. Some variants of SCMC genes have been linked to female infertility characterized by embryonic development arrest. However, so far, the candidate non-core SCMC components associated with embryonic development need further exploration and the pathogenic variants that have been identified are still limited. In this study, we discovered two novel variants [p.(Ala131Val) and p.(Met326Val)] of NLRP2 in patients with primary infertility displaying embryonic development arrest from large families. In vitro studies using 293T cells and mouse oocytes, respectively, showed that these variants significantly decreased protein expression and caused the phenotype of embryonic development arrest. Additionally, we combined the 'DevOmics' database with the whole exome sequence data of our cohort and screened out a new candidate non-core SCMC gene ZFP36L2. Its variants [p.(Ala241Pro) and p.(Pro291dup)] were found to be responsible for embryonic development arrest. Co-immunoprecipitation experiments in 293T cells, used to demonstrate the interaction between proteins, verified that ZFP36L2 is one of the human SCMC components, and microinjection of ZFP36L2 complementary RNA variants into mouse oocytes affected embryonic development. Furthermore, the ZFP36L2 variants were associated with disrupted stability of its target mRNAs, which resulted in aberrant H3K4me3 and H3K9me3 levels. These disruptions decreased oocyte quality and further developmental potential. Overall, this is the first report of ZFP36L2 as a non-core component of the human SCMC and we found four novel pathogenic variants in the NLRP2 and ZFP36L2 genes in 4 of 161 patients that caused human embryonic development arrest. These findings contribute to the genetic diagnosis of female infertility and provide new insights into the physiological function of SCMC in female reproduction.

Rui X ，Zhang X ，Jia X ，Han J ，Wang C ，Cao Q ，Zhong O ，Ding J ，Zhao C ，Zhang J ，Ling X ，Li H ，Ma X ，Meng Q ，Huo R ... -  《-》

NLRP7 participates in the human subcortical maternal complex and its variants cause female infertility characterized by early embryo arrest.

Successful human reproduction requires normal oocyte maturation, fertilization, and early embryo development. Early embryo arrest is a common phenomenon leading to female infertility, but the genetic basis is largely unknown. NLR family pyrin domain-containing 7 (NLRP7) is a member of the NLRP subfamily. Previous studies have shown that variants of NLRP7 are one of the crucial causes of female recurrent hydatidiform mole, but whether NLRP7 variants can directly affect early embryo development is unclear. We performed whole-exome sequencing in patients who experienced early embryo arrest, and five heterozygous variants (c.251G > A, c.1258G > A, c.1441G > A, c. 2227G > A, c.2323C > T) of NLRP7 were identified in affected individuals. Plasmids of NLRP7 and subcortical maternal complex components were overexpressed in 293 T cells, and Co-IP experiments showed that NLRP7 interacted with NLRP5, TLE6, PADI6, NLRP2, KHDC3L, OOEP, and ZBED3. Injecting complementary RNAs in mouse oocytes and early embryos showed that NLRP7 variants influenced the oocyte quality and some of the variants significantly affected early embryo development. These findings contribute to our understanding of the role of NLRP7 in human early embryo development and provide a new genetic marker for clinical early embryo arrest patients. KEY MESSAGES: Five heterozygous variants of NLRP7 (c.1441G > A; 2227G > A; c.251G > A; c.1258G > A; c.2323C > T) were identified in five infertile patients who experienced early embryo arrest. NLRP7 is a component of human subcortical maternal complex. NLRP7 variants lead to poor quality of oocytes and early embryo development arrest. This study provides a new genetic marker for clinical early embryo arrest patients.

Han J ，Zhang N ，Cao Q ，Shi X ，Wang C ，Rui X ，Ding J ，Zhao C ，Zhang J ，Ling X ，Li H ，Guan Y ，Meng Q ，Huo R ... -  《-》

Mutations in OOEP and NLRP5 identified in infertile patients with early embryonic arrest.

The subcortical maternal complex (SCMC), composed of several maternal-effect genes, is vital for the development of oocytes and early embryos. Variants of SCMC-encoding genes (NLRP2, NLRP5, TLE6, PADI6, and KHDC3L, but not OOEP and ZBED3) are associated with human oocyte maturation dysfunction, fertilization failure, and early embryonic arrest. In this study, we enrolled 118 Chinese patients who experienced recurrent preimplantation embryonic arrest during assisted reproductive technology treatments and performed whole-exome sequencing. We discovered compound heterozygous missense variants (c.110G>C and c.109C>G) in the OOEP gene in one patient who experienced recurrent preimplantation embryonic arrest. Arrested embryos from this affected patient were analyzed by single-cell RNA sequencing, which showed a downregulated transcriptome. In addition, six novel NLRP5 variants (c.971T>A, c.3341T>C, c.1575_1576delAG, c.1830_1831delGT, c.1202C>T, and c.2378T>G) were identified in four patients with arrested and severely fragmented embryos. These suspicious mutations were examined by in vitro studies in HEK293T cells. Western blot analysis and immunofluorescence experiments showed that OOEP and partial NLRP5 mutations caused decreased protein levels. Our findings first demonstrated that biallelic variants in OOEP gene could also cause human early embryonic arrest, similar to other SCMC components. We expanded the genetic mutation spectrum of SCMC genes related to early embryogenesis in humans, especially early embryonic arrest.

Tong X ，Jin J ，Hu Z ，Zhang Y ，Fan HY ，Zhang YL ，Zhang S ... -  《-》

Expanding the genetic and phenotypic spectrum of the subcortical maternal complex genes in recurrent preimplantation embryonic arrest.

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.

Zheng W ，Hu H ，Dai J ，Zhang S ，Gu Y ，Dai C ，Guo J ，Xu X ，Li Y ，Zhang S ，Hu L ，Gong F ，Lu G ，Lin G ... -  《-》

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. NCT03354013.

Cardona Barberán A ，Bonte D ，Boel A ，Thys V ，Paredis R ，Machtelinckx F ，De Sutter P ，De Croo I ，Leybaert L ，Stoop D ，Coucke P ，Vanden Meerschaut F ，Heindryckx B ... -  《-》