Sperm Selection Using Zona Pellucida-Binding Enhanced Embryo Morphokinetic and Clinical Outcomes in ICSI: A Sibling Oocytes Study.

The objective was to investigate the embryo morphokinitics using a time-lapse monitoring (TLM) system and assessment of clinical outcomes following intracytoplasmic sperm injection (ICSI) with zona pellucida (ZP)-bound sperm selection and conventional methods. A total of 371 metaphase II (MII) oocytes from 50 ICSI cycles were studied. Sibling oocytes were randomly divided into control (n = 199) and ZP-bound group (n = 172). All resulting zygotes were cultured and monitored in the TLM system up to Day 3 after ICSI. Fertilization rate, early embryo development, and clinical outcomes were evaluated. No significant differences were found in fertilization rate, time-lapse qualitative and quantitative measures, pronuclear fading time (PNF) t2, t3, t4, t5, t6, and t7 (times of cleavage to 2, 3, 4, 5, 6, and 7 cells), respectively. However, the t8 (time of cleavage to eight cells) and cc3 (duration of third cell cycle) revealed a significant difference between control and ZP-bound groups (p < .05). A significant difference between the two groups (p < .05) in the rates of Grade A embryos (according to Basile algorithm), chemical pregnancy, clinical pregnancy, and implantation was observed. Sperm selection using biological materials, such as ZP, improved both embryo quality and pregnancy outcomes, despite not affecting the early embryo development and morphokinetic parameters up to t8. This prospective randomized sibling oocyte trial was registered in October 2020 to January 2022 (IRCT20200705048021N1).

Xeno-free generation of new Yazd human embryonic stem cell lines (Yazd4-7) as a prior stage toward good manufacturing practice of clinical-grade raw materials from discarded embryos: A lab resources report.

Background: Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cell-based therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines. Objective: To produce new hESC lines in xeno-free condition. Materials and Methods: This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity. Results: In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer. Conclusion: The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practice-derived hESC derivatives.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

OBJECTIVE: Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). MATERIALS AND METHODS: In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. RESULTS: Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). CONCLUSION: Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

Defined Physicochemical Cues Steering Direct Neuronal Reprogramming on Colloidal Self-Assembled Patterns (cSAPs).

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Mesenchymal stem cells-derived exosomes as a promising new approach for the treatment of infertility caused by polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial metabolic and most common endocrine disorder that its prevalence, depending on different methods of evaluating PCOS traits, varies from 4% to 21%. Chronic low-grade inflammation and irregular apoptosis of granulosa cells play a crucial role in the pathogenesis of PCOS infertility. Mesenchymal stem cells (MSCs)-derived exosomes and extracellular vesicles (EVs) are lipid bilayer complexes that act as a means of intercellular transferring of proteins, lipids, DNA and different types of RNAs. It seems that this nanoparticles have therapeutic effects on the PCOS ovary such as regulating immunity response, anti-inflammatory (local and systemic) and suppress of granulosa cells (GCs) apoptosis. Although there are few studies demonstrating the effects of exosomes on PCOS and their exact mechanisms is still unknown, in the present study we reviewed the available studies of the functions of MSC-derived exosome, EVs and secretome on apoptosis of granulosa cells and inflammation in the ovary. Therefore, the novel cell-free therapeutic approaches for PCOS were suggested in this study.

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report.

Background: Fibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have wound-healing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells. Objective: Generation and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs). Materials and Methods: In this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flow-cytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45. Results: The YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics. Conclusion: This study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine.

Use of zona pellucida-bound spermatozoa as a natural selection in improvement of ICSI outcomes: A systematic review and meta-analysis.

Zona pellucida (ZP)-bound spermatozoa have normal morphology and motility and can enhance the ICSI outcomes. Selection of zona pellucida-bound spermatozoa is recently considered to find functional spermatozoa for ICSI. This study reviewed the efficacy of ZP-bound sperm selection on the ICSI outcomes includes fertilisation rate, embryo quality, embryo transfer rate and clinical pregnancy rate. The databases searched include PubMed, Scopus and Cochrane databases up to January 2019. All research reports with full text and in English language that addressing the relation between ZP-sperm selection and ICSI outcomes were included. Fifty studies were suitable after screening of the 845 identified articles. After exclusions, five of these studies were included. Meta-analytic pooling of data indicated no association between the ICSI outcomes and ZP-bound sperm selection except a marginal effect on implantation rate. Eliminating one study indicated that ZP-bound sperm selection technique improves embryo quality, implantation rate and clinical pregnancy rate. This study revealed that ZP-bound sperm selection produces only a slight improvement in implantation rate. However, further studies with a large number of couples must be done to clarify the potential beneficial effect of ZP-bound spermatozoa on ICSI outcomes.

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles.

Background: Human embryonic stem cell-mesenchymal stem/stromal cell (hESCs-MSCs) open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods: Herein, hESCs-MSCs were characterized by immunofluorescence technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using reverse transcription-polymerase chain reaction technique. Fluorescence-activated cell sorting was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results: MSCs were derived from diploid and triploid hESC lines using sequential three dimensional and two dimensional cultures and characterized with the specific markers. Immunofluorescence showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Cobnclusion: Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.

Potential of Mesenchymal Stem Cell-Derived Exosomes as a Novel Treatment for Female Infertility Caused by Bacterial Infections.

Neisseria gonorrhoeae and Chlamydia trachomatis are the most common causes of bacterial sexually transmitted diseases (STDs) with complications in women, including pelvic inflammatory disease (PID), ectopic pregnancy, and infertility. The main concern with these infections is that 70% of infected women are asymptomatic and these infections ascend to the upper female reproductive tract (FRT). Primary infection in epithelial cells creates a cascade of events that leads to secretion of pro-inflammatory cytokines that stimulate innate immunity. Production of various cytokines is damaging to mucosal barriers, and tissue destruction leads to ciliated epithelial destruction that is associated with tubal scarring and ultimately provides the conditions for infertility. Mesenchymal stem cells (MSCs) are known as tissue specific stem cells with limited self-renewal capacity and the ability to repair damaged tissues in a variety of pathological conditions due to their multipotential differentiation capacity. Moreover, MSCs secrete exosomes that contain bioactive factors such as proteins, lipids, chemokines, enzymes, cytokines, and immunomodulatory factors which have therapeutic properties to enhance recovery activity and modulate immune responses. Experimental studies have shown that local and systemic treatment of MSC-derived exosomes (MSC-Exos) suppresses the destructive immune response due to the delivery of immunomodulatory proteins. Interestingly, some recent data have indicated that MSC-Exos display strong antimicrobial effects, by the secretion of antimicrobial peptides and proteins (AMPs), and increase bacterial clearance by enhancing the phagocytic activity of host immune cells. Considering MSC-Exos can secrete different bioactive factors that can modulate the immune system and prevent infection, exosome therapy is considered as a new therapeutic method in the treatment of inflammatory and microbial diseases. Here we intend to review the possible application of MSC-Exos in female reproductive system bacterial diseases.

Mesenchymal Stem-Cell Derived Exosome Therapy as a Potential Future Approach for Treatment of Male Infertility Caused by Chlamydia Infection.

Some microbial sexually transmitted infections (STIs) have adverse effects on the reproductive tract, sperm function, and male fertility. Given that STIs are often asymptomatic and cause major complications such as urogenital inflammation, fibrosis, and scarring, optimal treatments should be performed to prevent the noxious effect of STIs on male fertility. Among STIs, Chlamydia trachomatis is the most common asymptomatic preventable bacterial STI. C. trachomatis can affect both sperm and the male reproductive tract. Recently, mesenchymal stem cells (MSCs) derived exosomes have been considered as a new therapeutic medicine due to their immunomodulatory, anti-inflammatory, anti-oxidant, and regenerative effects without consequences through the stem cell transplantation based therapies. Inflammation of the genital tract and sperm dysfunction are the consequences of the microbial infections, especially Chlamydia trachomatis. Exosome therapy as a noninvasive approach has shown promising results on the ability to regenerate the damaged sperm and treating asthenozoospermia. Recent experimental methods may be helpful in the novel treatments of male infertility. Thus, it is demonstrated that exosomes play an important role in preventing the consequences of infection, and thereby preventing inflammation, reducing cell damage, inhibiting fibrogenesis, and reducing scar formation. This review aimed to overview the studies about the potential therapeutic roles of MSCs-derived exosomes on sperm abnormalities and male infertility caused by STIs.

Characteristics of the human endometrial regeneration cells as a potential source for future stem cell-based therapies: A lab resources study.

BACKGROUND: Human endometrium with consecutive regeneration capability undergoes monthly hormonal changes for probable implantation, which confirms the presence of the cells in the basalis layer known as stem cell. OBJECTIVE: Previously, we reported the isolation and culture of the mesenchymal-like cells from human endometrium. In this study, we evaluated the biological and stemness characteristics of these cells. MATERIALS AND METHODS: The characterization of Yazd human endometrial-derived mesenchymal stem/stromal cells (YhEnMSCs) was assessed using immunofluorescence (IF) staining for CD105, VIMENTIN, and FIBRONECTIN as markers and RT-PCR for CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, MHCI, CD14, and MHCII genes. Flow cytometry (FACS) was performed for CD44, CD73, CD90, and CD105 markers. Moreover, the differentiation capacity of the YhEnMSCs to the osteoblast and adipocytes was confirmed by Alizarin Red and Oil Red staining. RESULTS: YhEnMSCs expressed CD105, VIMENTIN, FIBRONECTIN, CD44, CD73, and CD90 markers and CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, and MHCI, but, did not express CD14, MHCII. CONCLUSION: Our data confirm previous reports by other groups indicating the application of endometrial cells as an available source of MSCs with self-renewal and differentiation capacity. Accordingly, YhEnMSCs can be used as a suitable source for cell-based therapies.

Effect of Human Testicular Cells Conditioned Medium on In Vitro Maturation and Morphology of Mouse Oocytes.

BACKGROUND: Testicular cell conditioned medium (TCCM) has been shown to induce female germ cell development In Vitro from embryonic stem cells (ESCs). Testicular cells (TCs) secrete a variety of growth factors such as growth differentiation factor-9 (GDF-9), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), leukemia inhibitory factor (LIF), and other, that could improve oocyte maturation. Here we have investigated the effect of human TCCM (hTCCM) on in vitro maturation (IVM) and morphology of mouse oocytes. MATERIALS AND METHODS: In this experimental study, 360 germinal vesicle (GV) oocytes were obtained from NMRI mice, aged 4-6 weeks that had received 5 IU pregnant mare's serum gonadotropin (PMSG) 48 hours before. GV oocytes were subjected to IVM. 120 GV oocytes were cultured in each medium; hTCCM as the test group, DMEM + 20%FBS as the control group and Ham's F10 + HFF medium as the sham group. The rates of the IVM and perivitelline space (PVS) changes were recorded at 8, 16 and 24 hours after culture. The metaphase II (MII) oocytes were subjected for in vitro fertilization (IVF) and the fertilization rate was evaluated after 1, 2, and 3 days. RESULTS: There was a significant difference between the maturation rates in hTCCM (31.67% MII) and the control [0% MII, P<0.05, (7.5% MI, 52.5% deg. and 40%GV)] groups but there was not a significant difference between the maturation rates in hTCCM and the sham group (53.33% MII, P>0.05). IVF success rate for MII oocytes obtained from IVM in the hTCCM group was 28.94% (n=11). Our data showed that hTCCM is an effective medium for GV oocyte growth and maturation compared to the control medium. CONCLUSION: Our findings show that TCCM supports oocyte IVM in mice and affect oocyte morphology.

A comparative analysis of immunomodulatory genes in two clonal subpopulations of CD90+ amniocytes isolated from human amniotic fluid.

OBJECT: Immunosuppressive and immunomodulatory activity of mesenchymal stem cells derived from different sources, such as placental membranes, umbilical cord, and amniotic fluid has been proved. The heterogeneous nature of human amniocytes have been confirmed due to different clonal subpopulations found in amniotic fluid. The aim of this study was to investigate a 17-gene panel of immunomodulatory markers in two clonal subpopulations of CD90+ amniocytes, divided based on morphology into epithelioid and fibroblastoid cells. METHOD: Semi-quantitative RT-PCR was used to study the expression of the chosen genes. Flow cytometry analysis confirmed the non-hematopoietic mesenchymal origin of isolated cells, based on lacking the hematopoietic marker of CD31, and the presence of mesenchymal marker of CD90 (both on more than 90% of cells). RESULTS: Our results showed that besides growth characteristics, the two cell groups were different in expressional profile, so that, fibroblastoid clones displayed higher level of immunosuppression genes as well as mesenchymal surface marker of CD90 compared to epithelioid ones. Our previous investigation on these clones showed that epithelioid cells have a more potential to express the pluripotency genes. It seems there is an inverse relationship between genes associated with immunosuppression and pluripotency. CONCLUSION: Although many reports have been published regarding the immunosuppressive properties of fetal stem cells, but few studies to date have explained whether the stemness state of human amniocytes may affect their immunosuppressive potential. Further study on amniocytes, which often has self-renewal ability and high immunomodulatory potential, can help to understand the details of this relationship.

The Viability of Human Testis-Derived Cells on Human Serum Albumin-Based Scaffold as An Artificial Male Germ Cell Niche.

Azoospermia is one of the challenging disorders affecting couples who are afflicted with infertility. Human testisderived cells (hTCs) are suitable candidates for the initiation of in-vitro spermatogenesis for these types of patients. The current study aimed to assess the proliferation of hTCs through the cell culture on the three-dimensional (3D) porous scaffolds. Cells harvested from the testicular sperm extraction (TESE) samples of the azoospermic patients were cultured on the 3D porous scaffolds containing human serum albumin (HSA)/tri calcium phosphate nanoparticles (TCP NPs) for two weeks. The proliferation/viability of the cells was assessed using the MTT assay, along with H and E histological staining method. The MTT assay showed that hTCs could stay alive on this scaffold with 50 and 66.66% viability after 7 and 14 days, respectively. Such viability was not significantly different when compared with cells grown on monolayer flask culture (P>0.05). Therefore, 3D HSA/TCP NPs scaffolds could be used for the reconstitution of the artificial human somatic testicular niche for future applications in regenerative medicine for male infertility.

The effect of the human cumulus cells-conditioned medium on in vitro maturation of mouse oocyte: An experimental study.

BACKGROUND: To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply in vitro maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation. OBJECTIVE: To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology. MATERIALS AND METHODS: In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days. RESULTS: A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium. CONCLUSION: hCCCM supports oocyte in vitro growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space.

Development of An Artificial Male Germ Cell Niche Using Electrospun Poly Vinyl Alcohol/Human Serum Albumin/Gelatin Fibers.

OBJECTIVE: Recent achievements in stem cell biotechnology, nanotechnology and tissue engineering have led to development of novel approaches in regenerative medicine. Azoospermia is one of the challenging disorders of the reproductive system. Several efforts were made for isolation and culture of testis-derived stem cells to treat male infertility. However, tissue engineering is the best approach to mimic the three dimensional microenvironment of the testis in vitro. We investigated whether human testis-derived cells (hTCs) obtained by testicular sperm extraction (TESE) can be cultured on a homemade scaffold composed of electrospun nanofibers of homogeneous poly (vinyl alcohol)/human serum albumin/gelatin (PVA/HSA/gelatin). MATERIALS AND METHODS: In this experimental lab study, human TCs underwent two steps of enzymatic cell isolation and five culture passages. Nanofibrous scaffolds were characterized by scanning electron microscopy (SEM) and Fouriertransform infrared spectroscopy (FTIR). Attachment of cells onto the scaffold was shown by hematoxylin and eosin (H and E) staining and SEM. Cell viability study using MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide] assay was performed on days 7 and 14. RESULTS: Visualization by H and E staining and SEM indicated that hTCs were seeded on the scaffold. MTT test showed that the PVA/HSA/gelatin scaffold is not toxic for hTCs. CONCLUSION: It seems that this PVA/HSA/gelatin scaffold is supportive for growth of hTCs.

Attempts for Generation of Embryonic Stem Cells from Human Embryos Following In Vitro Embryo Twinning.

In vitro embryo twinning can be used to increase the number of the human embryos available for production of human embryonic stem cell (hESC) lines. The aim of this study was to generate hESCs following the production of the twin embryos by in vitro embryo splitting procedures. In total 21 chromosomally abnormal (three pronuclei) embryos underwent in vitro embryo twinning and were allowed to develop to the blastocyst stage. As a result, 42 twin embryos were obtained, of which 24 developed to blastocyst stage. Using micromanipulation technique, the zona-free blastocysts were recovered and plated onto mitotically inactivated Yazd human foreskin fibroblast (Batch18; YhFF#18) feeder layers in microdrops. After 3 to 5 days of blastocyst culture onto human foreskin fibroblast feeder layers, the hESC-like outgrowths were passaged onto new feeders in microdrops. The initial outgrowths of hESC-like cells were generated, and cells were proliferated, passaged, and some of them expressed hESC and trophoblastic markers; however, no cell lines were established. This might be due to the low cell number and poor quality of inner cell mass within these twin blastocysts. In vitro embryo twinning by increasing the number of the human embryos could be useful in the future for the generation of new pluripotent stem cell lines. However, the challenge remains to optimize the methods.

Biological and physiological characteristics of human cumulus cells in adherent culture condition.

BACKGROUND: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment. OBJECTIVE: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells. MATERIALS AND METHODS: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture. RESULTS: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium. CONCLUSION: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies.

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report.

BACKGROUND: Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. OBJECTIVE: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. MATERIALS AND METHODS: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. RESULTS: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. CONCLUSION: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

Fresh versus frozen embryo transfer after gonadotropin-releasing hormone agonist trigger in gonadotropin-releasing hormone antagonist cycles among high responder women: A randomized, multi-center study.

Background: The use of embryo cryopreservation excludes the possible detrimental effects of ovarian stimulation on the endometrium, and higher reproductive outcomes following this policy have been reported. Moreover, gonadotropin-releasing hormone agonist trigger in gonadotropin-releasing hormone (GnRH) antagonist cycles as a substitute for standard human chorionic gonadotropin trigger, minimizes the risk of ovarian hyperstimulation syndrome (OHSS) in fresh as well as frozen embryo transfer cycles (FET). Objective: To compare the reproductive outcomes and risk of OHSS in fresh vs frozen embryo transfer in high responder patients, undergoing in vitro fertilization triggered with a bolus of GnRH agonist. Materials and Methods: In this randomized, multi-centre study, 121 women undergoing FET and 119 women undergoing fresh ET were investigated as regards clinical pregnancy as the primary outcome and the chemical pregnancy, live birth, OHSS development, and perinatal data as secondary outcomes. Results: There were no significant differences between FET and fresh groups regarding chemical (46.4% vs. 40.2%, p=0.352), clinical (35.8% vs. 38.3%, p=0.699), and ongoing (30.3% vs. 32.7%, p=0.700) pregnancy rates, also live birth (30.3% vs. 29.9%, p=0.953), perinatal outcomes, and OHSS development (35.6% vs. 42.9%, p=0.337). No woman developed severe OHSS and no one required admission to hospital. Conclusion: Our findings suggest that GnRHa trigger followed by fresh transfer with modified luteal phase support in terms of a small human chorionic gonadotropin bolus is a good strategy to secure good live birth rates and a low risk of clinically relevant OHSS development in in vitro fertilization patients at risk of OHSS.