Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate: optimal follicle size for oocyte retrieval.

OBJECTIVE: To analyze oocyte competence in gonadotropin-releasing hormone agonist (GnRHa) stimulation cycles with regard to maturity, fertilization and blastocyst rate, as well as clinical outcome (pregnancy and live-birth rate), in relation to follicular volume, measured by three-dimensional transvaginal sonography (3D-TVS), and follicular fluid composition. METHODS: This was a prospective single-center study conducted between June 2012 and June 2014, including 118 ovum pick-ups with subsequent embryo transfer. Ovarian stimulation was performed using the GnRHa long protocol. Of 1493 follicles aspirated individually, follicular volume was evaluated successfully in 1236 using automated 3D-TVS during oocyte retrieval. Oocyte maturity and blastocyst development were tracked according to follicular volume. Intrafollicular concentrations of estradiol, testosterone, progesterone, luteinizing hormone, follicle-stimulating hormone and granulocyte-colony stimulating factor were quantified by immunoassay. Clinical outcome, in terms of implantation rate, (clinical) pregnancy rate, miscarriage and live-birth rate (LBR), was evaluated. RESULTS: Follicles were categorized, according to their volume, into three arbitrary groups, which included 196 small (8-12 mm/0.3-0.9 mL), 772 medium (13-23 mm/1-6 mL) and 268 large (≥ 24 mm/> 6 mL) follicles. Although oocyte recovery rate was significantly lower in small follicles compared with medium and large ones (63.8% vs 76.6% and 81.3%, respectively; P < 0.001), similar fertilization rates (85.1% vs 75.3% and 81.4%, respectively) and blastocyst rates (40.5% vs 40.6% and 37.2%, respectively) per mature metaphase II oocyte were observed. A trend towards higher LBR after transfer of blastocysts derived from small (< 1 mL) follicles compared with medium (1-6 mL) or large (> 6 mL) follicles (54.5% vs 42.0%, and 41.7%, respectively) was observed. No predictive value of follicular fluid biomarkers was identified. CONCLUSIONS: Our data indicate that the optimal follicular volume for a high yield of good quality blastocysts with good potential to lead to a live birth is 13-23 mm/1-6 mL. However, oocytes derived from small follicles (8-12 mm/0.3-0.9 mL) still have the capacity for normal development and subsequent delivery of healthy children, suggesting that aspiration of these follicles should be encouraged as this would increase the total number of blastocysts retrieved per stimulation. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

[How can the haematopoietic stem cells from the umbilical cord blood be de-differentiated in vitro? Our first results using the co-cultivation systems]. / Jak dediferencovat hematopoetické kmenové bunky z umbilikální krve in vitro? Nase první výsledky s pouzitím kokultivacních systému.

OBJECTIVE: Aim of this study was to de-differentiate the haematopoietic stem cells (HSCs) that originated from the umbilical cord blood. One of the ways to do it is to use a co-cultivation system. DESIGN: Prospective experimental study. SETTING: Laboratory study - Institute of reproductive medicine and endocrinology, Pilsen. METHODS: HSCs were co-cultivated with mouse embryonic stem cells (mESC) with and without feeder cells. After co-cultivation HSCs were analyzed using flow-cytometry for presence of haematopoietic markers (CD34, CD45, CD133) and using immunohistochemistry for presence of embryonic stem cell markers (SSEA-4, Tra-1-60, Tra-1-81). RESULTS: No de-differentiation was detectable in any our experiment, only the intensity of the HSC cell markers decreased. CONCLUSION: We suppose that there were two major reasons for the experiment failure: there was no direct cell to cell contact and there was a mixture of cell types that originated from two different species. To reach our goal of in vitro de-differentiation we will need to change our strategy towards a pure human culture system without any animal additives and with cell to cell contact.

Apoptosis affects integration frequency: adult stem cells injected in blastocysts show high caspase-3 activity.

Chimeric organisms are commonly generated by injecting stem cells into blastocysts. Embryonic stem cells injected into the blastocoel cavity participate in the further development of the embryo. Adult stem cells have also been used in injection experiments to study their potential plasticity. In this study we focused on the early fate of injected human adult hematopoietic stem cells (HSCs). HSCs were followed immunohistochemically 1-19 h after injection into murine blastocysts. We found that they only rarely attached and integrated into the blastocysts. The high rate of loss of injected cells after prolonged in vitro culture of the chimeras can be explained by apoptosis. Our findings are consistent with previous studies reporting a low rate of integration of adult cells injected to produce chimeric embryos, but this is the first demonstration that the low efficiency of adult stem cell injections into blastocysts is influenced by apoptosis.

GnRH agonist versus GnRH antagonist in oocyte donation cycles: a prospective randomized study.

BACKGROUND: The specific role of LH in folliculogenesis and oocyte maturation is unclear. GnRH antagonists, when administered in the late follicular phase, induce a sharp decrease in serum LH which may be detrimental for IVF outcome. This study was performed to evaluate whether the replacement of GnRH agonist (triptorelin) by a GnRH antagonist (ganirelix; NV Organon) in oocyte donation cycles has any impact on pregnancy and implantation rates. METHODS: A total of 148 donor IVF cycles was randomly assigned to use either a GnRH antagonist daily administered from the 8th day of stimulation (group I) or a GnRH agonist long protocol (group II) for the ovarian stimulation of their donors. The primary endpoints were the pregnancy and the implantation rates. RESULTS: The clinical pregnancy rate per transfer (39.72%, 29/73 versus 41.33%, 31/75) based on transvaginal scan findings at 7 weeks of gestation, the implantation rate (23.9 versus 25.4%) and the first trimester abortion rate (10.34 versus 12.90%) were similar in the two groups. CONCLUSION: In oocyte donation cycles the replacement of GnRH agonist by a GnRH antagonist appears to have no impact on the pregnancy and implantation rates when its administration starts on day 8 of stimulation.

Paraffin-embedded manipulated blastocysts: a tool to demonstrate stem cell plasticity?

One of the big question marks in current stem cell research is whether there is true plasticity of adult progenitor cells (APC) or if cell fusion is the principle source of the supposed plasticity. The generation of chimeras by injecting adult progenitor cells into blastocysts is not new. This paper describes an efficient embedding technique for murine blastocysts injected with human APC. This method could help in establishing a novel tool to analyse the process of plasticity, if it truly exists. If this is the case, this technology could be of great help to characterize surface markers of stem cells in great detail. On the other hand, fusion of cells could also be investigated. A system of embedding blastocysts was set up using paraffin for further analysis by means of light microscopy and immunohistochemistry. The embedding of the chimaeras consists of fixing them first with paraformaldehyde in phosphate-buffered saline (PFA/PBS), embedding them in gelatine, fixing the gelatine block with PFA/PBS and finally fixing the gelatine block in a Petri dish by embedding it in paraffin. Using this protocol, the morphology of the blastocysts is well preserved.

Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility.

BACKGROUND: The study aim was to evaluate the relationship between pronuclei morphology scoring (PNMS) and the chromosomal complement of embryos in couples with severe male infertility undergoing ICSI. A total of 3116 pre-embryos was scored according to PNMS in 452 cycles. METHODS: Pre-embryos were classified into eight categories based on the alignment, size, linear or irregular distribution of pronuclear bodies (PNB), position and clarity of cytoplasmic halo and abutting of the pronucleus. These categories were subdivided into groups I and II according to the similarity and distribution of PNB. RESULTS: In total, 2574 pre-embryos formed by using ejaculated sperm, while 542 pre-embryos developed by injection of testicular sperm or round spermatids. More group II pre-embryos with markedly different morphology from group I were formed after ICSI with testicular sperm than with fresh ejaculated sperm (32.1 versus 22.7%, P < 0.01). Of 490 pre-embryos in which pronuclear morphology was evaluated, 263 were biopsied for preimplantation genetic diagnosis. The rate of chromosomal abnormality was higher in embryos developed from group II pre-embryos (52.2%) than in embryos developed from group I prezygotes (37.6%, P < 0.05). CONCLUSIONS: Group II pre-embryos had markedly different morphology from group I, and had a low rate of blastocyst formation and high risk of chromosomally abnormal embryos. When testicular sperm and round spermatids were used for ICSI, more group II pre-embryos and chromosomally abnormal embryos were produced than with ejaculated sperm. Pronuclear morphology was correlated with chromosomal complement, and impacted upon by the sperm source.

[Medically assisted reproduction with immature spermatozoa. Clinical examination and surgical technics]. / La procréation médicalement assistée avec spermatozoïdes immatures. Examens cliniques et techniques chirurgicales.

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation.

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection-testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men.

In non-obstructive azoospermia spermatozoa can usually only be isolated from the testicles, and thus the most promising treatment model is testicular sperm extraction (TESE). Hormone concentrations, testicular volume determinations and testicular biopsy results are not uniform enough to select potential candidates for successful TESE and intracytoplasmic sperm injection (ICSI) approaches in advance. The aim of this study was to assess the efficacy of using ICSI with testicular spermatozoa in cases of non-obstructive azoospermia and to compare the inclusion criteria and sperm existence in the testicles in sperm obtainable and non-obtainable groups. All men showed either complete or incomplete (n = 14) maturation arrest in spermatogenesis, severe hypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n = 5) in their testicular biopsies. Only 14 out of a total of 29 men provided enough spermatozoa for the ICSI procedure, while no spermatozoa were found in the testicular samples of the remaining 15 men. Out of 123 oocytes obtained from 14 females, 101 were injected with the husbands' testicular sperm cells. Total fertilization failure was observed in three cases. Of 39 oocytes fertilized, 38 cleaved. The fertilization and cleavage rates were 38.6 and 97.4% respectively. The pregnancy rate was 20.7% per initiated cycle. In the group from whom spermatozoa were obtainable, the pregnancy rate was 42.9% per initiated cycle and 54.5% per embryo transfer. A total of six pregnancies were achieved, of which two were twins and four were singletons. One singleton pregnancy resulted in abortion in the first trimester. There was no statistical difference concerning the serum follicle stimulating hormone concentration, testicular volume and biopsy results in groups in which spermatozoa were obtainable or not. In conclusion, although the association of TESE with ICSI obtained pregnancies for some patients with non-obstructive azoospermia, further studies are needed to determine the inclusion criteria for successful TESE.

Intracytoplasmic sperm injection, fertilization, and embryo transfer after retrieval of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy.

OBJECTIVE: To achieve fertilization and cleavage by spermatozoa retrieved by testicular biopsy from a male with testicular tubular atrophy. DESIGN: Clinical trial. SETTING: Private reproductive institute. PATIENT: Azoospermic male with demonstrated testicular tubular atrophy and almost complete spermatogenic arrest. INTERVENTION: Open biopsy retrieval of testicular tissue and sperm followed by intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Fertilization and cleavage. RESULTS: One four- to six-cell embryo was formed after intracytoplasmic sperm injection of five eggs with extruded polar bodies by retrieved sperm. CONCLUSION: Intracytoplasmic sperm injection after testicular sperm aspiration may be attempted in cases with severely decreased spermatogenesis and result in fertilization, cleavage, and embryo transfer.

Usefulness of partial dissection of the zona pellucida in a human in-vitro fertilization programme.

The influence of partial zona dissection (PZD) on the fertilization rate was studied in 34 couples with a history of fertilization failure and/or severe sperm deficiency. Overall, PZD improved the rate of monospermic fertilization compared to controls (41/254 versus 6/111: P less than 0.001) and fertilization was achieved in 50% of cases. However, the results differed according to the seminal characteristics. In 10 couples with at least two in-vitro fertilization (IVF) trials entailing total fertilization failure and with semen defined as normal, PZD did not significantly improve the monospermic fertilization rate (6/44 in the PZD group versus 2/39 among controls). A benefit related to PZD was evident in 33 attempts with severe sperm deficiency. In this group, only four of 72 unmanipulated control oocytes were fertilized but the monospermic fertilization rate was 14.6% for PZD oocytes. The rates of polyspermy were 40% and 14.6% in the groups with normal and abnormal semen parameters respectively. Of 33 trials with defective spermatozoa, 20 reached the stage of embryo transfer and three pregnancies were obtained, of which one aborted at 9 weeks.