Evaluation of the Clinical Efficacy and Trust in AI-Assisted Embryo Ranking: Survey-Based Prospective Study.

BACKGROUND: Current embryo assessment methods for in vitro fertilization depend on subjective morphological assessments. Recently, artificial intelligence (AI) has emerged as a promising tool for embryo assessment; however, its clinical efficacy and trustworthiness remain unproven. Simulation studies may provide additional evidence, provided that they are meticulously designed to mitigate bias and variance. OBJECTIVE: The primary objective of this study was to evaluate the benefits of an AI model for predicting clinical pregnancy through well-designed simulations. The secondary objective was to identify the characteristics of and potential bias in the subgroups of embryologists with varying degrees of experience. METHODS: This simulation study involved a questionnaire-based survey conducted on 61 embryologists with varying levels of experience from 12 in vitro fertilization clinics. The survey was conducted via Google Forms (Google Inc) in three phases: (1) phase 1, an initial assessment (December 23, 2022, to January 22, 2023); (2) phase 2, a validation assessment (March 6, 2023, to April 5, 2023); and (3) phase 3 an AI-guided assessment (March 6, 2023, to April 5, 2023). Inter- and intraobserver assessments and the accuracy of embryo selection from 360 day-5 embryos before and after AI guidance were analyzed for all embryologists and subgroups of senior and junior embryologists. RESULTS: With AI guidance, the interobserver agreement increased from 0.355 to 0.527 and from 0.440 to 0.524 for junior and senior embryologists, respectively, thus reaching similar levels of agreement. In a test of accurate embryo selection with 90 questions, the numbers of correct responses by the embryologists only, embryologists with AI guidance, and AI only were 34 (38%), 45 (50%), and 59 (66%), respectively. Without AI, the average score (accuracy) of the junior group was 33.516 (37%), while that of the senior group was 35.967 (40%), with P<.001 in the t test. With AI guidance, the average score (accuracy) of the junior group increased to 46.581 (52%), reaching a level similar to that of the senior embryologists of 44.833 (50%), with P=.34. Junior embryologists had a higher level of trust in the AI score. CONCLUSIONS: This study demonstrates the potential benefits of AI in selecting embryos with high chances of pregnancy, particularly for embryologists with 5 years or less of experience, possibly due to their trust in AI. Thus, using AI as an auxiliary tool in clinical practice has the potential to improve embryo assessment and increase the probability of a successful pregnancy.

Improved prediction of clinical pregnancy using artificial intelligence with enhanced inner cell mass and trophectoderm images.

This study aimed to assess the performance of an artificial intelligence (AI) model for predicting clinical pregnancy using enhanced inner cell mass (ICM) and trophectoderm (TE) images. In this retrospective study, we included static images of 2555 day-5-blastocysts from seven in vitro fertilization centers in South Korea. The main outcome of the study was the predictive capability of the model to detect clinical pregnancies (gestational sac). Compared with the original embryo images, the use of enhanced ICM and TE images improved the average area under the receiver operating characteristic curve for the AI model from 0.716 to 0.741. Additionally, a gradient-weighted class activation mapping analysis demonstrated that the enhanced image-trained AI model was able to extract features from crucial areas of the embryo in 99% (506/512) of the cases. Particularly, it could extract the ICM and TE. In contrast, the AI model trained on the original images focused on the main areas in only 86% (438/512) of the cases. Our results highlight the potential efficacy of using ICM- and TE-enhanced embryo images when training AI models to predict clinical pregnancy.

Mitochondrial and Metabolic Dynamics of Endometrial Stromal Cells During the Endometrial Cycle.

The endometrial cycle in response to hormonal stimulation is essential for implantation. The female has endometrium that repeats this cycle through about half of a lifetime. The cycle includes three phases, proliferative, secretory, and menstrual, and each phase has distinct characteristics. The endometrial stromal cells (EnSCs) in each phase also have specialized characteristics, including cell cycle, morphologies, and cellular metabolic state. So we hypothesized that the cells in each phase have unique mitochondrial morphologies because they are generally linked to cellular metabolic state. To investigate the metabolic characteristics in each phase, we investigated the mitochondrial morphologies by transmission electron microscopy, oxygen consumption rate (OCR), and intracellular adenosine triphosphate (ATP) production. The decidualized EnSCs have shorter mitochondria than those in the proliferative phase. Besides, they also displayed distinct intracellular structural characteristics compared with the proliferative phase, such as ribosome-rich endoplasmic reticulum and increased formation of vesicles. OCR and luminescent ATP detection assay revealed that the basal respiration and ATP production in the decidualized EnSCs were lower than those in the proliferative phase. Thus, we concluded that morphological and intracellular structural changes were induced during the decidualization. Moreover, the decreased mitochondrial length was shown to correlate with decreased dependency on oxidative phosphorylation and ATP concentration in EnSCs.

Alterations in intracellular Ca2+ levels in human endometrial stromal cells after decidualization.

Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1 µM medroxyprogesterone acetate) and cyclic AMP (0.5 mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.

Mitochondrial and metabolic remodeling during reprogramming and differentiation of the reprogrammed cells.

Reprogramming is one of the most essential areas of research in stem cell biology. Despite this importance, the mechanism and correlates of reprogramming remain largely unknown. In this study, we investigated the cytoplasmic remodeling and changes in metabolism that occur during reprogramming and differentiation of pluripotent stem cells. Specifically, we examined the cellular organelles of three pluripotent stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and epiblast stem cells (EpiSCs), by electron microscopy. We found that the cellular organelles of primed pluripotent EpiSCs were more similar to those of naive pluripotent ESCs and iPSCs than somatic cells. EpiSCs, as well as ESCs and iPSCs, contain large nuclei, poorly developed endoplasmic reticula, and underdeveloped cristae; however, their mitochondria were still mature relative to the mitochondria of ESCs and iPSCs. Next, we differentiated these pluripotent stem cells into neural stem cells (NSCs) in vitro and compared the morphology of organelles. We found that the morphology of organelles of NSCs differentiated from ESCs, iPSCs, and EpiSCs was indistinguishable from brain-derived NSCs. Finally, we examined the changes in energy metabolism that accompanied mitochondrial remodeling during reprogramming and differentiation. We found that the glycolytic activity of ESCs and iPSCs was greater compared with EpiSCs, and that the glycolytic activity of EpiSCs was greater compared with NSCs differentiated from ESCs, iPSCs, and EpiSCs. These results suggest that a change in the cellular state is accompanied by dynamic changes in the morphology of cytoplasmic organelles and corresponding changes in energy metabolism.

Antioxidative phytoceuticals to ameliorate pancreatitis in animal models: an answer from nature.

Despite enthusiastic efforts directed at elucidating critical underlying mechanisms towards the identification of novel therapeutic targets for severe acute pancreatitis (SAP), the disease remains without a specific therapy to be executed within the first hours to days after onset of symptoms. Although earlier management for SAP should aim to either treat organ failure or reduce infectious complications, the current standard of care for the general management of AP in the first hours to days after onset of symptoms include intravenous fluid replacement, nutritional changes, and the use of analgesics with a close monitoring of vital signs. Furthermore, repeated evaluation of severity is very important, as the condition is particularly unstable in the early stages. In cases where biliary pancreatitis is accompanied by acute cholangitis or in cases where biliary stasis is suspected, an early endoscopic retrograde cholangiopancreatography is recommended. However, practice guidelines regarding the treatment of pancreatitis are suboptimal. In chronic pancreatitis, conservative management strategies include lifestyle modifications and dietary changes followed by analgesics and pancreatic enzyme supplementation. Recently, attention has been focused on phytoceuticals or antioxidants as agents that could surpass the limitations associated with currently available therapies. Because oxidative stress has been shown to play an important role in the pathogenesis of pancreatitis, antioxidants alone or combined with conventional therapy may improve oxidative-stress-induced organ damage. Interest in phytoceuticals stems from their potential use as simple, accurate tools for pancreatitis prognostication that could replace older and more tedious methods. Therefore, the use of antioxidative nutrition or phytoceuticals may represent a new direction for clinical research in pancreatitis. In this review article, recent advances in the understanding of the pathogenesis of pancreatitis are discussed and the paradigm shift underway to develop phytoceuticals and antioxidants to treat it is introduced. Despite the promise of studies evaluating the effects of antioxidants/phytoceuticals in pancreatitis, translation to the clinic has thus far been disappointing. However, it is expected that continued research will provide solid evidence to justify the use of antioxidative phytoceuticals in the treatment of pancreatitis.

Predictive proteomic biomarkers for inflammatory bowel disease-associated cancer: where are we now in the era of the next generation proteomics?

Recent advances in genomic medicine have opened up the possibility of tailored medicine that may eventually replace traditional "one-size-fits all" approaches to the treatment of inflammatory bowel disease (IBD). In addition to exploring the interactions between hosts and microbes, referred to as the microbiome, a variety of strategies that can be tailored to an individual in the coming era of personalized medicine in the treatment of IBD are being investigated. These include prompt genomic screening of patients at risk of developing IBD, the utility of molecular discrimination of IBD subtypes among patients diagnosed with IBD, and the discovery of proteome biomarkers to diagnose or predict cancer risks. Host genetic factors influence the etiology of IBD, as do microbial ecosystems in the human bowel, which are not uniform, but instead represent many different microhabitats that can be influenced by diet and might affect processes essential to bowel metabolism. Further advances in basic research regarding intestinal inflammation may reveal new insights into the role of inflammatory mediators, referred to as the inflammasome, and the macromolecular complex of metabolites formed by intestinal bacteria. Collectively, knowledge of the inflammasome and metagenomics will lead to the development of biomarkers for IBD that target specific pathogenic mechanisms involved in the spontaneous progress of IBD. In this review article, our recent results regarding the discovery of potential proteomic biomarkers using a label-free quantification technique are introduced and on-going projects contributing to either the discrimination of IBD subtypes or to the prediction of cancer risks are accompanied by updated information from IBD biomarker research.

Comprehensive chromosome analysis of blastocysts before implantation using array CGH.

BACKGROUND: Chromosomal abnormalities are common in embryos produced in vitro and cause implantation failure, miscarriage, and serious medical problems in infants. Because preimplantation genetic screening (PGS) is increasingly being used to detect aneuploidy in embryos with the purpose of improving implantation rates after IVF (in vitro fertilization), we aimed to validate the usefulness of array CGH for the preimplantation genetic screening (PGS) of embryos at the blastocyst stage of development. RESULTS: A total of 150 blastocysts were biopsied from couples undergoing IVF and analyzed using array CGH. We found that 54.5% (73/134) of the blastocysts were euploid embryos, whereas 45.5% of the embryos (61/134) had chromosomal abnormalities. Multiple chromosome abnormality was most frequently observed (34.4%), and dual aneuploidy was observed in 26.2% of the embryos. Monosomy (21.3%) appeared more frequently than trisomy (18%). CONCLUSION: Chromosomal microarray analysis provided clinically significant cytogenetic information regarding the frequency and variety of chromosomal abnormalities observed in embryos at the blastocyst stage, suggesting that this is a useful tool for comprehensive aneuploidy screening in IVF.

Apolipoprotein A-IV as a novel gene associated with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder, affecting 6-10% of women of reproductive age. The etiology remains poorly understood. To investigate the differentially expressed proteins from PCOS patients versus healthy women, the protein expression in follicular fluid was analyzed using two-dimensional electrophoresis (2-DE). Since follicular fluid contains a number of secretory proteins required for oocyte fertilization and follicle maturation, it is possible that follicular fluid can be used as a provisional source for identifying pivotal proteins associated with PCOS. In this study, six overexpressed proteins [kininogen 1, cytokeratin 9, antithrombin, fibrinogen γ-chain, apolipoprotein A-IV (apoA-IV) precursor and α-1-B-glycoprotein (A1BG)] in follicular fluids from PCOS patients were identified with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS. Western blot analysis confirmed that the protein expression levels of apoA-IV precursor and A1BG were increased in follicular fluid from PCOS patients compared with those from normal controls. The analysis of protein expression for other proteins revealed individual variation. These results facilitate the understanding of the molecular mechanisms of PCOS and provide candidate biomarkers for the development of diagnostic and therapeutic tools.

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture.

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.

Ethanol-induced small heat shock protein genes in the differentiation of mouse embryonic neural stem cells.

Neural stem cells (NSCs) of the neuroepithelium differentiate into one of three central nervous system (CNS) cell lineages: neurons, astrocytes, or oligodendrocytes. In this study, the differentiation potential of NSCs from the forebrain of embryonic day 15 (E15) mouse embryos was analyzed using immunocytochemistry. NSCs were differentiated early in the presence or absence of ethanol (50 mM), and gene expression patterns among NSCs, differentiated cells and ethanol-treated differentiated cells were assessed by microarray and real-time PCR analysis. Genes that were up-regulated in differentiated cells both in the presence and in the absence of ethanol when compared to NSCs were related to the Wnt signaling pathway, including Ctnna1, Wnt5a, Wnt5b, Wnt7a, Fzd3, and Fzd2; genes related to cell adhesion, including Cadm1, Ncam1, and Ncam2; and genes encoding small heat shock proteins, including HspB2, HspB7, and HspB8. In particular, the expression levels of HspB2 and HspB7 were elevated in ethanol-treated differentiated cells compared to non-treated differentiated cells. The gene expression patterns of various heat shock transcription factors (HSFs), proteins that regulate the transcription of heat shock genes, were also analyzed. The expression levels of HSF2 and HSF5 increased in differentiated cells in the presence and absence of ethanol when compared to NSCs. Of these two genes, HSF5 demonstrated an enhanced up-regulation, particularly in ethanol-treated differentiated cells compared to cells that were differentiated in the absence of ethanol. These results imply that HspB2 and HspB7, which are small heat shock proteins with tissue-restricted expression profiles, might be up-regulated by ethanol during the short-term differentiation of NSCs.

Effects of acute ethanol treatment on NCCIT cells and NCCIT cell-derived embryoid bodies (EBs).

This study used human embryonic carcinoma (NCCIT) cells to evaluate genotoxicity and other effects of ethanol in earlier stages of cellular development. This undifferentiated pluripotent cell line has unlimited self-renewal capacity and has been shown to differentiate in vitro. We analyzed proteome expression profile of ethanol-treated NCCIT cells and NCCIT cell-derived embryoid bodies (EBs) by MALDI-TOF MS. To test the role of ethanol as an embryotoxic and/or teratogenic factor, MetaCore pathway analysis software (GeneGO) was used to evaluate the process of normal growth and differentiation of NCCIT cells and EBs. We compared the different protein expression profiles of ethanol-treated versus untreated NCCIT cells and NCCIT cell-derived EBs. The ethanol-treated NCCIT cells demonstrate significant up regulation of SMAP1, dual specificity phosphatase 1 and pro isomerase domain-containing 1, cytokeratin 18, triosephosphate isomerase and beta-tubulin. However, ethanol-treated NCCIT cell-derived EBs exhibited upregulated signatures of different proteins, including CDC25B phosphatase, alpha-enolase, 3-phosphoglycerate dehydrogenase and tumor suppressor patched L' isoform, which suggests that ethanol may play a different role in EBs. These proteins exert their function on transcriptional and translational processes. Moreover, the functional proteomic analysis confirms the relationship between ethanol and ethanol-regulated genes and various signaling pathways and networks. The data presented in this study contribute toward the understanding of the molecular mechanisms of ethanol in NCCIT cells and NCCIT cell-derived EBs.

High expression of large-conductance Ca2+-activated K+ channel in the CD133+ subpopulation of SH-SY5Y neuroblastoma cells.

Solid tumors contain a population of cancer stem cells (CSCs), and CD133 is widely used as a CSCs marker. We investigated the differences between CD133(+) and CD133(-) cells from the neuroblastoma cell line SH-SY5Y in terms of the expressions of voltage-gated ion channels. A CD133(+) enriched (>60%) population was isolated, and a subsequent whole-cell voltage-clamp study showed that these cells predominantly express TEA-sensitive outward K(+) currents (I(K,TEA)) and TTX-sensitive voltage-gated inward Na(+) currents (I(Na)). Cell-attached single channel recordings demonstrated higher density of large-conductance (155pS) channel in CD133(+) cells than in CD133(-) cells. The TEA-sensitivity and single channel conductance indicated the large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). Furthermore, RT-PCR analysis of 22 transcripts of voltage-gated ion channels in SH-SY5Y cells showed the expressions of Cav1.3, Kir2.1, Kv1.4, Kv2.1, Kv4.2, Kv7.1, BK(Ca), and Nav1.7, and those of BK(Ca) and Nav1.7 were higher in CD133(+) than in CD133(-) cells. In addition, the increase of cytosolic Ca(2+) concentration ([Ca(2+)](c)) in response to ionomycin (a Ca(2+) ionophore) was higher and more sustained in CD133(+) than in CD133(-) cells. Plausibly membrane hyperpolarization via BK(Ca) might be responsible for the augmented Ca(2+) influx observed in CD133(+) cells. The physiological implications of the differential expression of BK(Ca) and Nav1.7 in CSCs require further investigation.

Artificial microRNA-based neurokinin-1 receptor gene silencing reduces alcohol consumption in mice.

In the brain, the stress system plays an important role in motivating continued alcohol use and relapse. The neuropeptide substance P and the neurokinin-1 receptor (NK1R) are involved in the stress response and drug reward systems. Recent findings have shown that the binding of ligands to NK1Rs decreases the self-administration of alcohol in mice. We examined the effect of an artificial microRNA (amiRNA) on the functional expression of NK1R in mouse brains. Lentiviruses expressing either an amiRNA targeting the NK1R (amiNK1R) or a negative control amiRNA (amiNC) were injected into mouse brains. Four weeks after amiRNA injection, we found that amiNK1R decreased the voluntary alcohol consumption compared to mice injected with amiNC. We also observed that NK1R expression was reduced in the hippocampus. RNA interference is an effective approach to regulate the expression of specific behavior-related genes. Our results support the potential use of amiRNA as a therapeutic agent for the treatment of alcohol dependence.

Human Hair Follicle Cells with the Cell Surface Marker CD34 Can Regenerate New Mouse Hair Follicles and Located in the Outer Root Sheath of Immunodeficient Nude Mice.

BACKGROUND AND OBJECTIVES: The bulge region of hair follicle has been reported as a putative reservoir of hair follicle stem cells. The purpose of this study was to compare hair follice CD34 negative (CD34-) cell with CD34 positive (CD34+) cell and to evaluate the ability to regenerate new hair of immunodeficient nude mouse. METHODS AND RESULTS: In this report, we isolated the cells with CD34, known as bulge-negative cell surface marker from cultured human hair follicle cells using by magnetic cell sorting (MACS), injected the cells to immunodeficient nude mouse. To determine immunological characterization, human hair follicle CD34+ cells and CD34- cells were assessed by flow cytometry. The localization of injected-CD34+ cells was assessed on formalin-fixed, paraffin-embedded mouse skin samples by in situ hybridization technique. Our findings show that the human hair follicle cells with cell surface marker CD34 were located in the outer root sheath of nude mouse after transplantation and the cells were able to regenerate new hair follicle in immunodeficient nude mouse. CD34- cells also were able to regenerate follicles in the mouse, however, CD34+ cells were able to regenerate much more hair follicle than CD34- cells. CONCLUSIONS: Therefore, the results of this study add new insight into the investigation of CD34 stem cell-related molecule in human hair follicles and suggest that not all human hair follicle stem cells reside in bulge region, but in a lager niche.

Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization-embryo transfer.

OBJECTIVE: To assess the obstetric outcome of pregnancies resulting from in vitro maturation (IVM) and IVF-ET of immature oocytes retrieved from women with polycystic ovary syndrome (PCOS). DESIGN: Prospective observational study. SETTING: University fertility clinic. PATIENT(S): One hundred thirty-nine women undergoing 203 IVM treatment cycles. INTERVENTION(S): Immature oocyte recovery from unstimulated ovaries. In vitro oocyte maturation and fertilization. Fresh ET and assessment of obstetric outcomes in the pregnant women. MAIN OUTCOME MEASURE(S): Pregnancy and obstetric outcome. RESULT(S): Forty-one pregnancies were obtained in 187 ETs, resulting in a pregnancy rate of 21.9%. Except for three patients lost to follow-up in these pregnancies, the abortion and live birth rates were 36.8% (14 of 38) and 63.2% (24 of 38), respectively. The mean (+/-SD) gestational age and birth weight at delivery for singletons were 38.4 +/- 2.0 weeks (range, 33-41.6 weeks) and 3,252 +/- 516 g (1,750-4,100 g), respectively. For twins these were 36.7 +/- 1.9 weeks (34.6-39 weeks) and 2,361 +/- 304 g (1,900-2,990 g), respectively. Pregnancy complications occurred in five patients (13.2%); these included preterm labor (n = 3) and placenta previa (n = 2). Two patients (5.3%) had a major congenital anomaly diagnosed by ultrasonography. CONCLUSION(S): The abortion rate, gestational age and birth weight at delivery, and obstetric complications of pregnancies conceived by IVM-ET in women with PCOS were comparable with those of other women with PCOS being treated by conventional IVF-ET. In vitro maturation followed by IVF-ET seems to be a useful treatment option for women with PCOS, thus avoiding the risk of ovarian hyperstimulation syndrome.

Successful spouse pregnancy of male patients with severe aplastic anemia and chronic myelogenous leukemia using spermatozoa banked prior to bone marrow transplantation and using the ICSI procedure: case reports.

PURPOSE: To report two cases of successful spouse pregnancies which were conceived with long-term cryopreserved spermatozoa that had been collected prior to the male patients receiving a bone marrow transplant. METHODS: The first case is the pregnant wife of a 25-year-old man with chronic myelogenous leukemia, whose semen was collected before bone marrow transplant and then cryopreserved, thawed, and then injected into the wife's eggs via ICSI. The second case is a 28-year-old man with severe aplastic anemia who became a father after his wife's eggs were fertilized via ICSI with thawed spermatozoa. RESULTS: These two cases were achieved pregnancies. CONCLUSIONS: These cases support research that men with malignancy have the chance of fathering their own genetic children. Therefore, it is important to increase the awareness of clinicians, oncologists, and patients to the new developments in preserving fertility for cancer patients.