The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts -recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.

Context. Anti-Mullerian hormone (AMH) assay is becoming the best indicator of successful IVF treatment response to fertility drugs and could be a useful marker of embryo implantation potential. Various protocols are being used for controlled ovarian stimulation (COS), but there is an uncertainty regarding the implementation of the best protocol for endometriosis patients and also little evidence is available concerning the clinical value of AMH levels in endometriosis.Objective. This study aimed to evaluate the prognostic value of serum AMH levels for pregnancy in COS using GnRH-agonist(GnRH-a) and GnRHantagonist(GnRH-ant) protocols in endometriosis patients.Design. This is a cross-sectional study between March 2012 and November 2015.Subjects and Methods. Data were collected from 249 COS cycles of endometriosis patients, including 129 cycles with GnRH-a and 120 cycles with GnRH-ant. Patients in each group were classified into three subgroups based on their serum AMH levels. The outcomes of ICSI program were evaluated.Results. The ROC curve analysis showed that embryo and oocyte counts and AMH were equally predictive for pregnancy, as demonstrated by a similar area under the curve (AUC) of 0.69, 0.66 and 0.64, respectively. The sensitivity and specificity for prediction of positive pregnancy were 70.91% and 67.01% for embryo counts, 70.91% and 67.53% for oocyte counts at the cutoff values of 5 and 7, respectively, and 83.64% and 52.58% for AMH levels at the cutoff values of 1.3ng/mL.Conclusions. This study demonstrates that AMH as a single test has substantial accuracy in the prediction of pregnancy using the GnRH antagonist protocol for patients with endometriosis. In other words, AMH assay prior to ovarian stimulation initiation guides the clinicians to choose the antagonist stimulation protocol for the patients with two extreme AMH levels. AMH levels can be used to individualize control ovarian stimulation in endometriosis patients.

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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.

Sperm selection is a clinical need for guided fertilization in men with low-quality semen. In this regard, microfluidics can provide an enabling platform for the precise manipulation and separation of high-quality sperm cells through applying various stimuli, including chemical agents, mechanical forces, and thermal gradients. In addition, microfluidic platforms can help to guide sperms and oocytes for controlled in vitro fertilization or sperm sorting using both passive and active methods. Herein, we present a detailed review of the use of various microfluidic methods for sorting and categorizing sperms for different applications. The advantages and disadvantages of each method are further discussed and future perspectives in the field are given.