Background. It is widely assumed that the uterine cavity in non-pregnant women is physiologically sterile, also as a premise to the long-held view that human infants develop in a sterile uterine ...environment, though likely reflecting under-appraisal of the extent of the human bacterial metacommunity. In an exploratory study, we aimed to investigate the putative presence of a uterine microbiome in a selected series of non-pregnant women through deep sequencing of the V1-2 hypervariable region of the 16S ribosomal RNA (rRNA) gene. Methods. Nineteen women with various reproductive conditions, including subfertility, scheduled for hysteroscopy and not showing uterine anomalies were recruited. Subjects were highly diverse with regard to demographic and medical history and included nulliparous and parous women. Endometrial tissue and mucus harvesting was performed by use of a transcervical device designed to obtain endometrial biopsy, while avoiding cervicovaginal contamination. Bacteria were targeted by use of a barcoded Illumina MiSeq paired-end sequencing method targeting the 16S rRNA gene V1-2 region, yielding an average of 41,194 reads per sample after quality filtering. Taxonomic annotation was pursued by comparison with sequences available through the Ribosomal Database Project and the NCBI database. Results. Out of 183 unique 16S rRNA gene amplicon sequences, 15 phylotypes were present in all samples. In some 90% of the women included, community architecture was fairly similar inasmuch B. xylanisolvens, B. thetaiotaomicron, B. fragilis and an undetermined Pelomonas taxon constituted over one third of the endometrial bacterial community. On the singular phylotype level, six women showed predominance of L. crispatus or L. iners in the presence of the Bacteroides core. Two endometrial communities were highly dissimilar, largely lacking the Bacteroides core, one dominated by L. crispatus and another consisting of a highly diverse community, including Prevotella spp., Atopobium vaginae, and Mobiluncus curtisii. Discussion. Our findings are, albeit not necessarily generalizable, consistent with the presence of a unique microbiota dominated by Bacteroides residing on the endometrium of the human non-pregnant uterus. The transcervical sampling approach may be influenced to an unknown extent by endocervical microbiota, which remain uncharacterised, and therefore warrants further validation. Nonetheless, consistent with our understanding of the human microbiome, the uterine microbiota are likely to have a previously unrecognized role in uterine physiology and human reproduction. Further study is therefore warranted to document community ecology and dynamics of the uterine microbiota, as well as the role of the uterine microbiome in health and disease.
To assess the effect of two assisted oocyte activation (AOA) protocols with the use of two calcium (Ca(2+)) ionophores, ionomycin and A23187 (calcimycin), on the intracellular Ca(2+) level in mouse ...and human oocytes and the fertilization rates.
Comparison of two Ca(2+) ionophores, ionomycin and A23187, regarding their capacity to increase the intracellular Ca(2+) level and to support subsequent oocyte activation and development.
University hospital research laboratory.
Patients undergoing intracytoplasmic sperm injection (ICSI) treatment and B6D2F1 mice.
Assisted oocyte activation and microinjection of mouse and human oocytes with sperm.
Measurement of the fertilizing and Ca(2+)-releasing ability of human sperm.
Ionomycin was more potent than A23187 in provoking Ca(2+) increases in both mouse and human oocytes with significantly higher amplitude and area under the receiver operating characteristic curve. The oocyte activation rate was significantly higher when mouse oocytes were activated with the use of the ionomycin- rather than the A23187-based AOA protocol. Furthermore, oocyte activation rate was higher when human in vitro matured oocytes were activated with the ionomycin-based AOA protocol, but the difference did not reach statistical significance.
In both mouse and human oocytes, the AOA protocol that used ionomycin was more efficient than the one that used A23187. Bearing in mind that mammalian fertilization is successful when the total dose of Ca(2+) released reaches a minimal threshold, the use of ionomycin for human AOA might be justified instead of the use of A23187.
The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the ...events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.
OBJECTIVE: To examine the underlying factors leading to infertility in a male patient from whom phospholipase C zeta H398P (PLCζᴴ³⁹⁸ᴾ, histidine > proline) and PLCζᴴ²³³ᴸ (histidine > leucine) ...mutations were previously identified. DESIGN: Laboratory-based study. SETTING: University laboratory. PATIENT(S): An infertile 38-year-old man with significantly impaired oocyte activation ability. INTERVENTION(S): Minisequencing of individual sperm for PLCζᴴ³⁹⁸ᴾ and PLCζᴴ²³³ᴸ, and investigation of localization patterns arising from the expression of fluorescently tagged PLCζ isoforms in HEK293T cells. MAIN OUTCOME MEASURE(S): The presence/absence of PLCζᴴ³⁹⁸ᴾ and PLCζᴴ²³³ᴸ determined in individual sperm (n = 12 sperm), and localization of fluorescent mutant PLCζ isoforms quantified in HEK293T cells. RESULT(S): Sperm possessed either PLCζᴴ²³³ᴸ or PLCζᴴ³⁹⁸ᴾ, but never both at the same time. Fluorescent PLCζᴴ²³³ᴸ and PLCζᴴ²³³ᴸ⁺ᴴ³⁹⁸ᴾ (both mutations together) localized to discrete regions in HEK293T cytoplasm but not the plasma membrane. Fluorescence statistically significantly varied between constructs such that PLCζᵂᵀ > mutant isoforms at both 48- and 56-hour time points. Fluorescent-PLCζᴴ²³³ᴸ⁺ᴴ³⁹⁸ᴾ exhibited a statistically significantly reduced level of fluorescence compared with PLCζᴴ³⁹⁸ᴾ at 48 hours but not 56 hours. CONCLUSION(S): Both H398P and H233L mutations are present on different alleles and do not alter PLCζ localization in HEK293T cells. Loss-of-activity mutations in PLCζ may contribute not only toward male infertility but also male subfertility in cases where PLCζ is mutated on a single allele.
One consequence of the legal diversity in Europe is that legal restrictions on treatments can be evaded by going abroad. Many French lesbian couples and single women are crossing the border to ...Belgium because they are denied access to treatments with donor sperm at home. This is the first qualitative research study into the experiences and moral perspectives of these women. Between June 2012 and May 2013, 11 lesbian couples and 2 single women were recruited at the department of reproductive medicine at Ghent University Hospital. The data from the semi-structured interviews was analysed using inductive thematic analysis. The results show that these women face several additional challenges to the already difficult process of cross-border treatment. Before they can start the treatment, they can only obtain information from the internet or from stories of friends who also went abroad for treatment with donor sperm. During the treatment, they need to find local clinics or physicians to monitor their cycle. Several women managed to game the French system to ensure partial reimbursement for their treatment when they were successful in finding a physician who was willing to prescribe drugs and perform tests. Most women had difficulties justifying their absence from work. In general these women felt that they were discriminated against and that their rights were not protected because of who they are. In that regard, the lack of legal recognition of the genetically unrelated partner in their country was particularly hard to cope with for the lesbian couples. These women have to develop many different strategies to deal with the difficulties they face during cross-border reproductive care. It is concluded that it is very important that they find a physician who is willing to support them in their 'baby project'.
To evaluate the dose–response relationship of a novel recombinant human FSH (rhFSH; FE 999049) with respect to ovarian response in patients undergoing IVF/intracytoplasmic sperm injection treatment; ...and prospectively study the influence of initial antimüllerian hormone (AMH) concentrations.
Randomized, controlled, assessor-blinded, AMH-stratified (low: 5.0–14.9 pmol/L 0.7–<2.1 ng/mL; high: 15.0–44.9 pmol/L 2.1–6.3 ng/mL) trial.
Seven infertility centers in four countries.
Two hundred sixty-five women aged ≤37 years.
Controlled ovarian stimulation with either 5.2, 6.9, 8.6, 10.3, or 12.1 μg of rhFSH, or 11 μg (150 IU) of follitropin alfa in a GnRH antagonist cycle.
Number of oocytes retrieved.
The number of oocytes retrieved increased in an rhFSH dose–dependent manner, from 5.2 ± 3.3 oocytes with 5.2 μg/d to 12.2 ± 5.9 with 12.1 μg/d. The slopes of the rhFSH dose–response curves differed significantly between the two AMH strata, demonstrating that a 10% increase in dose resulted in 0.5 (95% confidence interval 0.2–0.7) and 1.0 (95% confidence interval 0.7–1.3) more oocytes in the low and high AMH stratum, respectively. Fertilization rate and blastocyst/oocyte ratio decreased significantly with increasing rhFSH doses in both AMH strata. No linear relationship was observed between rhFSH dose and number of blastocysts overall or by AMH strata. Five cases of ovarian hyperstimulation syndrome were reported for the three highest rhFSH doses and in the high AMH stratum.
Increasing rhFSH doses results in a linear increase in number of oocytes retrieved in an AMH-dependent manner. The availability of blastocysts is less influenced by the rhFSH dose and AMH level.
NCT01426386.
To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation ...genetic diagnosis (PGD) in translocation carriers.
Study of diagnostic accuracy.
University hospital.
Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation.
Trophectoderm biopsy was performed on 47 blastocysts.
In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar.
In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼ 4.5 Mb deletion/duplication.
This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.
Protocols for specifying human primordial germ cell‐like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and ...maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post‐inner cell mass intermediate PICMI) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i‐state improves differentiation to (TNAP tissue nonspecific alkaline phosphatase+/PDPN podoplanin+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP‐2 gamma), SOX17 (SRY‐box transcription factor 17), and NANOS3 (nanos C2HC‐type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C‐X‐C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i‐state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.
Human embryonic stem cells (hESCs) derived and cultured in the presence of activin A show higher propensity to differentiate to (TNAP+PDPN+) human primordial germ cell‐like cells, particularly when transiently converted to the 4i‐state prior to differentiation using an embryoid body aggregation assay.
Abstract The capacity of intracytoplasmic sperm injection (ICSI) to permit almost any type of spermatozoa to fertilize oocytes has made it the most successful treatment for male factor infertility. ...Despite its high success rates, fertilization failure following ICSI still occurs in 1–3% of couples. Assisted oocyte activation (AOA) is being increasingly applied in human assisted reproduction to restore fertilization and pregnancy rates in couples with a history of ICSI fertilization failure. However, controversy still exists mainly because the artificial activating agents do not mimic precisely the initial physiological processes of mammalian oocyte activation, which has led to safety concerns. This review addresses the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, it describes the current diagnostic approaches and focuses on the application, efficiency and safety of AOA in human assisted reproduction. Intracytoplasmic sperm injection (ICSI) is a very efficient technique used in couples diagnosed with severe sperm abnormalities. Fortunately, in only a minority of the couples ICSI does not lead to normal fertilization, which means that, in those couples, no (or very few) embryos will be available for transfer. Failure of fertilization following conventional ICSI can be related to the oocyte or the spermatozoon. Only a few diagnostic tests are currently available to assess the reason for ICSI fertilization failure. The most often advised treatment is assisted oocyte activation (AOA), a highly specialized fertilization technique that can be added to conventional ICSI to overcome fertilization failure in those couples. Since the late 1990s, many centres have been performing AOA in this rare group of patients, with restoration of fertilization and pregnancy rates in many couples. However, until adequate scientific evidence is provided regarding its safety and efficacy, AOA cannot yet be considered an established treatment. This review tackles the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, we describe the current diagnostic approaches and focus on the application, efficiency and safety of AOA in human assisted reproduction.
To investigate the effect of hormonal androgenic treatment on antimüllerian hormone (AMH) serum levels in female-to-male (FtM) transsexuals. Polycystic ovary syndrome (PCOS) is associated with ...elevated AMH levels. Some hypothesize that the high AMH level is a consequence of androgen-induced excessive development of small antral follicles. However, this role of androgens is not yet clear.
Observational, prospective, cohort study.
Tertiary academic medical center.
Twenty-two FtM transsexuals, healthy native females receiving cross-sex hormone therapy/androgenic treatment.
Androgenic treatment with testosterone (T) and an aromatase inhibitor while endogenous hormone secretion was suppressed with the use of a GnRH agonist.
Hormone concentrations were measured before and after androgenic treatment (administration of T and aromatase inhibitor). Measured hormones: AMH, inhibin B, T, androstenedione, DHEAS, E2, SHBG, LH, and FSH.
AMH concentrations were significantly lower after androgenic treatment (4.4 ± 4.4 μg/L vs. 1.4 ± 2.1 μg/L). Androgenic treatment resulted in a strong suppression of AMH secretion over a relative short period of 16 weeks.
Our data underscore the likely important role of androgens in the dynamics of folliculogenesis. It challenges the idea that androgens induce high AMH levels, which is gaining more interest nowadays as an important particular PCOS feature. This strong decline furthermore indicates that AMH must be interpreted in the context of other reproductive endocrine conditions.
NTR2493.
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