L. Gianaroli

The sperm centriole: its inheritance, replication and perpetuation in early human embryos

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

Incidence of Chromosomal Abnormalities from a Morphologically Normal Cohort of Embryos in Poor-Prognosis Patients

Purpose:Preimplantation genetic diagnosis of aneuploidy was performed on the embryos yielded by 70 poor-prognosis patients, with the aim of transferring those with a normal chromosomal complement, thus possibly increasing the chances of pregnancy.Methods:Multicolor fluorescence in situ hybridization (FISH) was applied for the simultaneous detection of chromosomes X, Y, 13, 16, 18, and 21. Inclusion criteria were (1) a maternal age of 36 years or older (n = 33), (2) three or more previous in vitro fertilization cycles (n = 20), and (3) an altered karyotype (n = 17).Results:A total of 412 embryos underwent FISH, resulting in 234 (57%) that were chromosomally abnormal. Euploid embryos were available for transfer in 59 patients, generating 19 pregnancies (32%), with an implantation rate of 19.9%.Conclusions:High rates of chromosomally abnormal embryos in poor-prognosis patients can determine repeated in vitro fertilization failures when embryo selection is performed on the basis of morphological criteria alone. Hence, the FISH analysis could represent the prevailing approach for the identification of embryos possessing full potential for developing to term.

Predicting aneuploidy in human oocytes: key factors which affect the meiotic process

BACKGROUND To estimate the incidence of aneuploidy in relation to patients characteristics, the type of hormonal stimulation and their response to induction of multiple follicular growth, 4163 first polar bodies (PB1s) were analyzed. METHODS Five hundred and forty four infertile couples underwent 706 assisted conception cycles (640 with poor prognosis indications and 66 controls) in which chromosomal analysis of PB1 for the chromosomes 13, 15, 16, 18, 21 and 22 was performed. Results were evaluated in a multivariate analysis. RESULTS The proportion of normal oocytes was directly correlated (P < 0.01) with (i) the number of mature oocytes and (ii) the establishment of a clinical pregnancy; and inversely correlated (P < 0.01) with (i) female age, (ii) causes of female infertility (endometriosis, abortions, ovulatory factor), (iii) poor prognosis indications (female age, number of previous cycles, multiple poor prognosis indications), (iv) number of FSH units per oocyte and (v) number of FSH units per metaphase II oocyte. There was a weak significance of frequency (P < 0.05) between type of abnormality (originated by chromatid predivision, chromosome non-disjunction or combined mechanisms in the same oocyte) and groups of the studied variables, rather than to a specific abnormality or a specific chromosome. CONCLUSIONS The type of infertility had a significant effect on errors derived from the first meiotic division, whose incidence was significantly higher in the presence of endometriosis or of an ovulatory factor, and in women that experienced repeated abortions. Each aneuploidy event was found to be dependent not on a specific variable, but on groups of variables. In addition, the tendency of chromosomal abnormalities to occur simultaneously implies that the deriving aneuploidies can be of any type.

Early human pregnancy in vitro utilizing an artificially perfused uterus

The penetration of luminal epithelium in the uterine cavity represents the crucial event that triggers the failure of embryo implant, thus limiting the possibility of fertility control. The purpose of our study was to implant a human blastocyst, cultured in vitro, into a human uterus extracorporeally perfused with an oxygenated medium. For this purpose, human blastocysts, collected from patients who underwent IVF program because of irreparable tubal infertility, were injected under the luminal epithelium of human perfused uteri. Light and electron microscopy showed that human blastocyst can successfully undergo the stage of implantation and trophoblastic invasion in 52 hours of extracorporeal perfusion.

Microinsemination techniques for the treatment of patients suffering agenesia of the vas deferens.

Thirty cycles of microsurgical epididymal sperm aspiration were performed on 27 patients presenting agenesia of the vas deferens. Two techniques of microinsemination were used, depending on the quality of the sperm preparation: subzonal sperm microinjection and microdroplet insemination. The results obtained by both microinsemination procedures are presented and discussed.

Sperm: Oocyte ratios in an in vitro fertilization (IVF) program

PurposeDuring the past few years, many oocyte insemination techniques, including microinjection, have evolved in the treatment of male-factor infertility. This preliminary study was designed to evaluate whether microdroplet insemination could be considered a reliable technique, especially for semen samples with male-factor defects. The first objective was to assess fertilization rates obtained by inseminating sibling oocytes using both the conventional IVF and the microdroplet method (Group 1). The second objective was to evaluate subsequent embryo development and pregnancy rates resulting from microdroplet insemination, in addition to formulating adequate sperm∶ oocyte ratios for various semen categories (Group 2). Four semen categories were studied including fresh normal sperm, frozen/thawed normal sperm, and male-factor sperm with one defect and two or three defects.ResultsGroup 1 consisted of 54 couples; no statistical significance was found in the fertilization rates between test tube and microdroplet insemination in all four semen categories. Based on these results, patients from Group 2 (48 couples) had their oocytes inseminated only in microdroplets with sperm:oocyte ratios ranging from 2000 to 10,000 motile sperm:1 oocyte. The average fertilization rate for male-factor sperm was 55%, with a 91% cleavage rate.ConclusionHigher fertilization rates were observed in the lowest range of sperm∶oocyte ratios (2000–4000∶1) for male-factor sperm with one defect and in the highest range (8000–10,000∶1) for male-factor sperm with two or three defects. Polyspermy occurred in only 0.4% of the oocytes inseminated. Microdroplet insemination is an alternative treatment for moderate to moderately severe male-factor infertility, establishing a bridge between conventional IVF and microinjection. With adequate sperm∶oocyte ratios, this technique allows the natural selection process of fertilization in vitro to take place, without the high incidence of polyspermy or mechanical damage frequently observed in assisted fertilization techniques.

Human amniotic fluid for fertilization and culture of human embryos: results of clinical trials in human in vitro fertilization (IVF) programs

We report the outcome of clinical trials carried out in two IVF programs, comparing the use of human amniotic fluid (HAF) as a complete medium to Whittinghams T6 medium containing human serum T6+10% HS) for egg incubation, insemination, embryo culture, and embryo transfer. There were no significant differences in the clinical trials between HAF used alone as a complete medium and T6+10% HS in fertilization rates of eggs, cleavage rates of embryos up to 48 hours in culture, pregnancy success rates after embryo replacement or the outcome of pregnancies. There was no advantage in using T6+10% HS for fertilization of eggs and HAF as a complete medium for embryo culture and transfer in any of the parameters examined. We conclude that HAF does not meet the complete requirements of human eggs and embryos in vitro and further developments of culture media are required to obtain embryo development equivalent to that in vivo.

Oxidative Stress Evaluation in Sperm Samples from Fertile and Infertile Men

Background: Transition metal ions, such as iron, can make electron donations to oxygen forming superoxide or hydrogen peroxide, which is further reduced to an extremely reactive hydroxyl radical that induces oxidative stress. The purpose of the present study was to design a system that could easily detect and reliably measure the ferrous oxidation associated to oxygen radical reactions in the sperm samples. Methods: A total of 64 sperm samples from 11 men who had normal semen parameters and proven fertility and 53 male partners of couple experiencing primary infertility, were included in the study. The semen samples from oligoasthenoteratozoospermic patients was divided on the basis of spermatic parameters into moderate, when the sperm concentration was ≥5 × 106/ml and in severe when the concentration was <5 × 106/ml. The evaluation of the ferrous oxidation was performed measuring the formation of iron complexes between ferric ions and thiocyanate anions by spectrofluorimetry. Results: The concentration of the ferric thiocyanate complex ions was significantly higher in pathological sperm samples (137.6 ± 10.8 μmol/l in moderate oligoasthenoteratozoospermic, 170.0 ± 25.4 μmol/l in severe oligoasthenoteratozoospermic and 155.4 ± 7.3 μmol/l in non-obtructive azoospermic men), when compared with both infertile noormozoospermic (92.4 ± 10.7 μmol/l) (P<0.015) and with samples from fertile men (76.3 ± 6.2 μmol/l) (P<0.005). No significant differences were found in the concentration of ferric thiocyanate complex among the different pathological groups when compared to each other and in infertile noormozoospermic patients when compared with the samples from men of proven fertility (P=0.168). Accordingly, an inverse correlation was found between the concentration of the ferric thiocyanate complex and total motility, progressive motility and morphology. Conclusions: This preliminary study shows that the method proposed detect quickly and reliably measures the ferrous oxidation associated to oxygen radical reactions in the sperm samples.

Subzonal sperm microinjection in cases of severe male factor infertility and repeated in vitro fertilization failure

OBJECTIVEnTo examine (1) fertilization rates obtained with subzonal sperm microinjection when different numbers of sperm are injected into the perivitelline space; (2) when subzonal sperm microinjection is combined with dilute insemination; and (3) the association of semen quality characteristics with fertilization.nnnDESIGNnSubzonal sperm microinjection and subzonal sperm microinjection combined with dilute insemination was performed in 109 and 41 cycles on patients in two clinical trials in Melbourne, Australia, and Bologna, Italy, respectively. PATIENT PARTICIPANTS: Couples who have experienced repeated in vitro fertilization failure or in whom the husband has severe male factor infertility.nnnPRIMARY OUTCOME MEASURESnThe number of oocytes fertilized after injection of different numbers of sperm into the perivitelline space, the number of patients transferred, and pregnancy outcome.nnnRESULTSnThe injection of multiple numbers of sperm into the perivitelline space failed to improve monospermic fertilization rates but caused an increase in polyspermic fertilization. In patients with initial semen parameters exhibiting greater than 50% motility or greater than 50% normal morphology fertilization rates were improved when subzonal sperm microinjection-treated eggs were incubated in a dilute insemination medium. Six pregnancies were obtained, two of which have progressed to term.nnnCONCLUSIONSnWhen applied to male factor patients, the subzonal sperm microinjection technique results in a 14% to 15% fertilization rate. However, of the 102 embryos transferred only three (2.9%) fetal heart beats were obtained.

Dr. Web and the New Generation of ART Patients

Over the last two decades, the role of the Internet and of related technologies has become preponderant in several areas of people’s lives and healthcare is undoubtedly one of these.