Seminal plasma is a complex mixture of secretions from various glands in the male genital tract. Compared to sperm cells, it contains important proteins that are both directly and indirectly associated with sperm motility. Here, we constructed quantitative proteomes of human seminal plasma from three normozoospermic and asthenozoospermic individuals. A total of 524 proteins were identified, and 366 of them were found to be quantified in all six samples. We first investigated the absolute expression features of these proteins and found that the variations of protein identification among different samples and other published datasets were mainly due to some lowly expressed proteins. By integration of various proteomic datasets and bioinformatics databases, we comprehensively annotated the biological functions, physiological originations, and disease associations of these proteins. We found that our dataset could benefit the studies of both male infertility and other male diseases. Finally, based on the relative expression values determined by chemical labeling, we identified a total of 29 differentially expressed proteins, which could be used as candidate targets for studying the molecular bases of sperm motility or developing precise diagnostic biomarkers of asthenozoospermia. We further successfully verified the expression trends of four representative proteins by Western blotting. Compared to a previous dataset based on label-free quantification, our results showed that most of the important proteins could be identified in the sample collected only once for each individual, providing the bases for personalized examination of seminal plasma proteins in clinic.

Epigenetic regulation, particularly post-translational modifications (PTMs) of histones, participates in spermatogonial stem cell (SSCs) differentiation. However, there is a lack of systemic studies of histone PTM regulation during the differentiation of SSCs due to its low number in vivo. Herein, we quantified dynamic changes of 46 different PTMs on histone H3.1 by targeted quantitative proteomics using mass spectrometry during SSCs differentiation in vitro, in combination with our RNA-seq data. We identified seven histone H3.1 modifications to be differentially regulated. In addition, we selected H3K9me2 and H3S10ph for subsequent biotinylated peptide pull-down experiments and identified 38 H3K9me2-binding proteins and 42 H3S10ph-binding proteins, which contain several transcription factors, such as GTF2E2 and SUPT5H, which appear to be crucial for epigenetic regulation of SSC differentiation.

What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2^Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2^Mut/Mut mice were verified at two months of age. Round spermatids from the littermates of wild-type (WT) and Adad2^Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2^WT/Mut male mice and six Adad2^WT/Mut female mice. A total of 120 Adad2^Mut/Mut, Adad2^WT/Mut and WT mice were used in this study. The entire study was conducted over three years. Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, Western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2^Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. Three recessive mutations were identified in ADAD2 (MT1: c. G829T, p. G277C; MT2: c. G1192A, p. D398N; MT3: c.917_918del, p. Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2^Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2^Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2^Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2^Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2^WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. N/A. This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2^Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2^Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. This work was supported by the National Natural Science Foundation of China (32000587, U21A20204 and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests. Non-obstructive azoospermia (NOA) is characterized by impaired spermatogenesis and is the most severe form of male infertility. Approximately 25% of NOA cases are attributed to genetic anomalies, but only mutations in a small number of genes have been validated as pathogenic in NOA patients. Our study focused on six infertile patients with NOA who were carriers of three ADAD2 mutations from different and unrelated families. Functional evidence has shown that these mutations cause premature degradation of the ADAD2 protein and are associated with failure to of round spermatids to differentiate into spermatozoa during spermatogenesis. Despite the lack of spermatozoa in the testes, Adad2^Mut/Mut mice with mutation efficiencies similar to that of human patients were able to produce healthy and fertile offspring after round spermatid injection. Therefore, our study provides a preliminary insight for the genetic counselling of couples with ADAD2-mutation-associated male infertility.

Embryo loss and pregnancy disorders are prevalent worldwide, with both conditions critically associated with dysfunctioning gastrulation processes. Gastrulation and post-gastrulation organogenesis are crucial stages of embryonic development that establish the blueprint for body part formation. These processes involve the sequential generation of three germ layer cells and primordial germ cells, as well as the assembly of the precursor tissues for body parts. However, due to ethical limitations associated with studying human embryogenesis, a more detailed understanding of gastrulation and post-gastrulation organogenesis remains elusive. To ensure that the knowledge obtained from gastruloids is biologically meaningful and clinically relevant, it is critical to create high-fidelity human embryo models that closely mimic embryogenesis in vivo . Here, we developed a two-stage derivation gastruloids in vitro based on human pluripotent stem cells. Morphological tracking mimicks the developmental processes of models from Carnegie Stage 4 (CS4) to early CS7. Our gastruloids exhibit key structures characteristic of human embryos, including amniotic cavity, embryonic disc, primitive streak, primary yolk sac, secondary yolk sac, and blood islets. Comparison of our cell lineage development maps showed that gastruloids closely resembled human natural CS7 gastrula. Our gastruloids exhibited transcriptional characteristics that mimicked the molecular pathways observed in natural embryos development. Importantly, we found that in our model, extraembryonic mesoderm originates from the yolk sac and primordial germ cells originate from the posterior epiblast of the embryonic disc. Moreover, we found that thalidomide affects the differentiation of three germ layer cells, resulting in the arrest of human gastruloid development. In conclusion, by establishing a human gastruloid, we were able to gain valuable insights into the mechanisms responsible for human gastrulation and shed light on the causes of early embryo loss and pregnancy disorders.

We study the risk factors that affect the formation of striae gravidarum in women in Jiangsu Province of China under current living habits. This sample is based on 400 primiparas from obstetric of the Affiliated Hospital of Jiangnan University in Wuxi from February 2017 to October 2017. Among them, there are 200 pregnant women with striae gravidarum and 200 without striae gravidarum. Striae gravidarum predictors were selected such as age, height, pre-pregnancy BMI, postpartum BMI, daily sitting time, weight gain during pregnancy, fetal weight, and diet on maternal. (1) The pregnant women who had striae gravidarum were generally lower in height than those who did not have striae gravidarum and had a higher BMI index than those who did not have striae gravidarum before and after childbirth. (2) In the pregnant women who have had striae gravidarum, the incidence of abdominal striae gravidarum in sedentary women is significantly lower than those in non-sedentary women, the incidence of striae gravidarum in legs is higher than those in non-sedentary women, and no significant difference in hip striae gravidarum. (3) In all pregnant women who have striae gravidarum, abdomen striae gravidarum tend to be lighter and leg striae gravidarum tends to be heavier in sedentary women, but no significant effect on the hip. (4) Regular consumption of honey, milk, trotters, freshwater fish, eggs, and tremella can reduce the incidence of striae gravidarum. The lifestyle and eating habits have a certain influence on the formation and severity of stretch marks.