Transcriptional Differences in Identical Twins With Different Reproductive Capacities: A Case Report

Disorders of sperm production can be classified quantitatively as oligospermia (low sperm count) or azoospermia (no sperm during ejaculation). Numerous genes have been implicated in spermatogenesis. We describe a case of two identical twins who presented with different reproductive capabilities. One brother was infertile due to azoospermia, and the other, although oligospermic, previously naturally fathered a child. They were found to have differential gene expression based on RNA sequencing analysis. In the man with azoospermia, we found elevated E2F1 and HOXB9 gene expressions when compared with his brother, suggesting that the increased RNA expression of these genes could influence sperm production.


Introduction
Male factor infertility has been found to be solely responsible for 20-30% of infertile couples and contributes to 50% of cases overall [1]. Qualitative and quantitative alterations in sperm parameters are possible, with quantitative disturbances classified based on severity. Azoospermia (no sperm present during ejaculation) has been identified in about 15% of infertile men and can be classified as obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) [2]. More than 2,000 genes are involved in spermatogenesis; therefore, the pathophysiology underlying disorders of spermatogenesis represents a highly complex interplay between genetic alterations and environmental aberrations [3]. Mutations in numerous genes have been implicated in the development of various abnormalities in sperm development [4,5]. Based on prior animal studies, numerous genes have been identified that play a role in spermatogenesis; E2F1 and HOXB9 are thought to regulate the G1/S transition and gene transcription, respectively [6,7].
In addition to these genetic causes, the epigenetic control of gene expression, often resulting from environmental or lifestyle factors, also plays a role in the development of male infertility, but it remains poorly understood [8]. In this study, we assessed differences in the RNA expression between two genetically identical individuals with different reproductive phenotypes. We examined the transcriptome in a unique case of identical twins, one with oligospermia and the other with NOA. Here, we present the first report of a novel finding of alteration in gene expression between genetically identical siblings. This work, previously presented at 78th Scientific Congress of the American Society for Reproductive Medicine, on October 25, 2022, presents a unique case description of the post-transcriptional modification of gene expression leading to phenotypic changes.

Case Presentation Patient selection
A 38-year-old Black male was identified as having NOA (testis volume of 10cc bilaterally and follicle stimulating hormone (FSH) of 14 IU/mL) during a routine male fertility evaluation. His twin brother was found to be oligospermic but had previously naturally fathered a child. Both patients denied personal history of organic cause of male infertility (testosterone use, infections, or testicular trauma). Both patients gave their consent to participate in the study. Institutional review board (IRB) approval (#20150740) was obtained from the University of Miami, Florida, United States.
Peripheral whole blood samples were collected. Total RNA was isolated using the TRIzol method. Next, reverse transcription to complementary DNA was induced using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA) according to the manufacturer's protocol.

RNA sequencing
RNA sequencing was completed at the John P Hussmann Institute for Human Genomics at the University of Miami. FastQ files were put through the FastQC program (Babraham Institute, UK) to check for quality. The adapter trimming software Trimgalore (Babraham Institute, UK), which uses the cutadaptpython package, was used for trimming. Reads were aligned against the human genome assembly GRCh37 (hg19) genome with the STAR RNAseq aligner and quantified against the GENCODE v19 database. Post-alignment quality control was performed with Picard tools (Broad Institute, USA) to check for ribosomal contented transcriptome alignment percentages. After alignment, differential expression comparisons were made with the DESeq2 R package (Bioconductor Project, USA). Due to the lack of replicates, the results from the comparison included nominal p-value significance (>0.05) instead of false discovery rate (FDR)-corrected pvalues, as well as normalized expression values. The normalized expression values were then put through a custom pipeline to determine patterns. First, minimum, maximum, and mean values were calculated from the four separate samples for each gene. Then, a standard deviation was calculated across the samples.

RNA analysis
A range of concentration expression values was formed using a maximum value of the baseline +0.75X standard deviation and a minimum value of the baseline -0.75X standard deviation. Different standard deviations were tested to check specificity and sensitivity levels, and 0.75X was determined to give the most viable results. The samples were lined up according to the concentration from zero to two. Comparisons to the baseline concentration classifications were calculated based on the comparison of the expression value to the calculated range. Those that were greater than the maximum range value were determined to be an upward change of expression, and those that were less than the minimum range value were determined to be a downregulation expression change in a given sample. Based on these patterns of change, we calculated the patterns that were of most clinical interest. Ingenuity Pathway Analysis (QIAGEN, USA) was used to enrich the selected markers from each section with respect to their involvement in molecular signaling networks.

RNA Analysis Results
Among the RNA sequencing results, we focused on protein coding genes with a fold change of greater than 10 times, resulting in 65 genes with 10-fold increase in expression and 132 with 10-fold decrease in expression (see Appendix). The resulting genes were subjected to enrichment using Ingenuity Pathway Analysis to identify the signaling pathways with which these candidates could be involved in. Ingenuity Pathway Analysis was utilized because it provides a robust platform for accurate gene expression analysis with a high degree of reproducibility. Importantly, the highlighted genes are involved in cell cycle regulation, which have previously been implicated in spermatogenesis. After comparing the RNAs of the brothers, we identified two genes differentially expressed in the NOA brother when compared to the oligospermic one. E2F1 expression was increased by 18-fold and HOXB9 expression by 11-fold in the NOA brother.

Discussion
We identified two monozygotic twins with defects in sperm production, one of them lacking sperm production. To understand the biological difference underlying their phenotypes, we focused on determining differences in gene expression, and we identified differential RNA expression in these genetically identical patients. We found increased of E2F1 and HOXB9 expressions in the azoospermic subject when compared to his oligospermic brother, which could represent post-transcriptional modification or epigenetic changes. E2F1 and HOXB9 are genes that play a role in the regulation of the cell cycle. E2F1 is a transcription factor that regulates heat shock protein 70 to control the G1/S checkpoint and assists with DNA repair, while HOXB9 regulates gene transcription through binding at a promoter region [6,7].
HOX genes are commonly associated with embryonic development and axonal positioning, as well as cell growth and regulation into adulthood [9,10]. HOXB9 is highly expressed in apoptotic cell lines and those with decreased proliferation and migration [11]. HOXB9 is expressed in T1 prospermatogonia-like cells [5].
Recently, HOXB9 was identified as a candidate gene responsible for male infertility in silico using cytogenetic data from cultured peripheral blood lymphocytes of infertile patients [12]. Based on these findings, the increase in HOXB9 expression in our NOA subject suggests that HOX activation may play a role in his defectivespermatogenesis.
E2F1 is a transcription factor regulator of spermatogenesis. E2F1 knockout mice showed decreased spermatogenesis that worsened through their lifetime culminating in azoospermia, testicular atrophy, and infertility [13,14]. Similarly, in mice, when transgenic overexpression of E2F1 was induced, testicular apoptosis was seen, resulting in azoospermia and infertility [15]. More importantly, microduplication and microdeletions of E2F1 have been linked to human cases of NOA in up to 7% of patients [16]. Rocca et al. confirmed these findings by reporting an association between NOA and E2F1 expression and hypothesizing that the copy number variants of E2F1 increase susceptibility to heat stress, thus, potentially, contributing to impairment in spermatogenesis [17]. Due to the impacts of E2F1 on germ cell survival and spermatogenesis, our finding of E2F1 upregulation in the azoospermic subject is of clinical importance.
Our study is not without limitations. Importantly, as a case series with two participants, the sample size is very small. With this RNA analysis, we are unable to assess the modification of the expression of these genes or the mechanisms behind the RNA expression changes. While Ingenuity Pathway Analysis provides an extensive, curated database of gene interactions and molecular pathways to aid in the interpretation of the biological context of our dataset, a significant quantitative difference in gene expression (as is seen in our study) is not solely indicative of pathology. This is particularly true when considering normal alleles, even when heterozygous, as differences in expression can occur naturally without signifying disease. While the differences exhibited in our study are notable and warrant further investigation, the changes in expression, in isolation, do not necessarily constitute proof of pathology. For example, these expression variants could be a part of a compensatory mechanism or an unrelated physiological process. Further functional studies are needed to definitively attribute these gene expression changes to the condition under investigation.
Despite these limitations, this study poses a unique opportunity to analyze the epigenetics of two genetically identical but phenotypically different men. These results contribute to the body of work characterizing the genes involved in sperm development. The identification of genes and their posttranscriptional modifications that play a role in spermatogenesis, such as HOXB9 and E2F1, provides further insights into the molecular mechanisms underlying sperm development. This case suggests that while genetic variations have been implicated in abnormalities of sperm development and function, epigenetic control of gene expression (which may be influenced by environmental or lifestyle factors) also plays a role in male fertility. Gene therapy targeting these specific genes may potentially have future implications on the management of infertile men. We hope that works at our institution and others can further elucidate the genes and mechanisms by which genetic variants influence sperm phenotypes.

Conclusions
This work presents a unique opportunity to assess differences in RNA expression in identical twins. We identified differences in HOXB9 and E2F1 RNA expression in identical twins with differential reproductive capacities, suggesting that these genes may impact spermatogenesis. Our results recapitulated the results of prior studies, indicating that increased expression in E2F1 cause azoospermia and infertility. Understanding the epigenetic basis of infertility is an important step in developing new treatments and interventions.  In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.