Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

MTHFR 1298A>C Substitution is a Strong Candidate for Analysis in Recurrent Pregnancy Loss: Evidence from 14,289 Subjects.

We undertook meta-analyses on MTHFR 1298A>C substitution for critically evaluating its association with recurrent pregnancy loss (RPL). MTHFR genotype data for 5888 cases and 8401 controls from 39 studies were pooled to perform this meta-analyses. Genotype data were screened, scrutinized, pooled, analysed and subjected to sensitivity analysis to carefully evaluate the association between MTHFR 1298A>C and recurrent pregnancy loss. Genetic associations were sought using dominant, recessive and co-dominant models of genetic testing with odds ratio and 95% Confidence interval (CI) as the effect measures. Further analyses were undertaken by classifying the studies into Caucasian and East Asian sub-groups. Genetic heterogeneity was tested before pooling the data across studies. For assessing publication bias, Egger's intercept test was undertaken. We found a significant association of 1298A>C substitution with increased risk of RPL in the dominant (P=0.000; OR = 1.58; 95% CI =1.25-1.99) as well as recessive (P=0.000; OR = 1.66; 95% CI =1.25-2.20) models. In sub-group analysis, we observed a significant association of the polymorphism with RPL in the Caucasian populations using dominant (P=0.000; OR = 1.98; 95% CI =1.42-2.76) and recessive (P=0.000; OR = 2.20; 95% CI =1.49-3.24) models. However, this substitution showed no association with RPL in the East Asian populations (P=0.149; OR = 1.187; 95% CI =0.94-1.50). MTHFR 1298A>C substitution shows association with the risk of recurrent pregnancy loss. The association is in a population-specific manner with the substitution being a strong risk factor only in the Caucasian populations.

Could SARS-CoV-2 affect male fertility?

We performed this systematic review to evaluate the possibility of an impact of SARS-CoV-2 infection on male fertility. SARS-CoV-2 enters the cells with the help of ACE2; therefore, testicular expression of ACE2 was analysed from transcriptome sequencing studies and our unpublished data. Literature suggested that SARS-CoV-1 (2002-2004 SARS) had a significant adverse impact on testicular architecture, suggesting a high possibility of the impact of SARS-CoV-2 as well. Out of two studies on semen samples from COVID-19 affected patients, one reported the presence of SARS-CoV-2 in the semen samples while the other denied it, raising conflict about its presence in the semen samples and the possibility of sexual transmission. Our transcriptome sequencing studies on rat testicular germ cells showed ACE expression in rat testicular germ cells. We also found ACE2 expression in transcriptome sequencing data for human spermatozoa, corroborating its presence in the testicular germ cells. Transcriptome sequencing data from literature search revealed ACE2 expression in the germ, Sertoli and Leydig cells. The presence of ACE2 on almost all testicular cells and the report of a significant impact of previous SARS coronavirus on testes suggest that SARS-CoV-2 is highly likely to affect testicular tissue, semen parameters and male fertility.

Coenzyme Q10 effect on semen parameters: Profound or meagre?

Coenzyme Q10 has shown promise in treating male infertility; however, there are inconsistencies across the published data. We undertook a quantitative meta-analysis by pooling data from three placebo-controlled randomised clinical trials (RCTs) in order to evaluate the efficacy of CoQ10 in improving semen parameters. Sperm count, sperm motility, sperm forward motility, sperm morphology and CoQ10 level in the seminal plasma were measured and quantitatively correlated with CoQ10 oral administration. Pooled analysis showed a significant impact of CoQ10 in improving sperm motility and forward motility, without a significant impact on sperm count, sperm morphology, ejaculate volume or seminal plasma level of CoQ10. Efficacy assessment suggested that CoQ10 shows better results at higher doses and when administered for a period of more than 3 months but not longer than 6 months. We conclude that CoQ10 has a profound effect on sperm motility and a meagre effect on all other parameters. Therefore, CoQ10 can be used for treating asthenozoospermic infertility with the dosage and duration depending upon the severity of the disorder and the patient's response to the treatment.

The dynamics of gene expression during and post meiosis sets the sperm agenda.

Meiosis is the defining event of spermatogenesis. Spermatocytes undergo meiosis to give rise to round spermatids, which in turn metamorphose to flagellated spermatozoa that mature in the epididymis. To characterize the dynamics of gene expression during these important stages of spermatogenesis, we undertook transcriptome analysis in >90% pure pachytene spermatocytes and round spermatids, and pure mature sperm of rat by massive parallel deep sequencing. The study has identified 10,719 total transcripts expressed in meiotic and postmeiotic cells, out of which 7,641 were present in all the three cell types. Most abundant transcripts were related to gametogenesis in spermatocytes and spermatids, and mitochondrial energy metabolism in sperm. Importantly, 108 transcripts were specific to spermatocytes, including Cpeb2, Dpf3, H2afy, Haus7, Plcb1, Taf9, and Tdrd7 strongly linked with meiosis. Similarly, 323 transcripts unique to round spermatids included Arpc5, Apoa1, Cntrob, Dcaf17, Ift88, and Ly6k that play essential roles in spermiogenesis. Likewise, 178 transcripts unique to sperm included Camta1, Hoxb1, and Prdx6 having assigned roles in fertility and/or embryonic development. Levels of ~16% transcripts declined from spermatocytes to sperm while two (Cd300e and Ddx17) increased. New candidate genes with possible roles in meiosis (91), spermiogenesis (298), and sperm function (171), have been identified. This study has provided new potential targets for contraception and/or treatment of male infertility. (CDRI communication number 9889).

The thermo-sensitive gene expression signatures of spermatogenesis.

BACKGROUND: Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment. METHODS: Testes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs. RESULTS: Heat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types. CONCLUSION: The study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.