LETTER Dear Editor-in-Chief: The Editorial of this year’s second issue of your interesting journal commented on the increasing use of time-lapse (TL) image systems in laboratories of assisted reproduction (Franco Jr, 2015). It was concluded that possible advantages of TL systems over classical morphological analyses are still lacking clear evidence. Moreover, new parameters assessing the pronuclear stage obviously have been introduced quite recently whereas the classical TL parameters mostly focused on the kinetics of embryo development (Franco Jr, 2015). Particularly with respect to deviations from the normal number of pronuclei (PN) I should like to add some thoughts. First, the formation of a supernumerary pronucleus is relatively common. However, whereas three (or even more) PN of approximately the same size may easily be recognized, there are cases with two normal-sized PN that are accompanied by an additional, rather small pronucleus. The basic mechanisms responsible for this phenomenon have been proposed earlier: either an incomplete chromatid segregation into the second polar body (PB) or an dispersal of the oocyte chromatids in the presence of a normal second PB (Rosenbusch, 2010). Because such abnormalities lead to irregular chromosome distributions into the blastomeres, pronuclear stages are carefully checked at 200x magnification in our laboratory and affected oocytes are excluded from transfer or cryopreservation. It has been noted that parameters associated with pronuclear morphology and dynamics may not be clearly observable with a dark-field TL system and that multiple planes will be necessary if the oocyte is not rotated (Ciray et al., 2014). Accordingly, the appropriate system has to be selected with great care if a precise inspection of the pronuclear constitution is envisaged. Of course, one could think of giving up the early PN assessment in favour of detecting aneuploidy at later embryonic stages since there are indications that aneuploid and normal embryos differ in the initiation of compaction and blastulation (Campbell et al., 2013) or, more generally, in their kinetic behaviour (Basile et al., 2014). On the other hand, it is questionable whether the latter approach that involves discarding (presumably aneuploid) cleavage stages will be an option in countries with strict embryo protection laws. Second, the appearance of a single pronucleus is another undesired event IVF laboratories have to deal with. As recently discussed (Rosenbusch, 2014), some embryos obtained from monopronuclear oocytes are biparental diploid and might be considered for transfer. How can the unexpected diploid constitution be explained? A single diploid pronucleus could arise when the paternal and maternal genomes are immediately enclosed in a common pronuclear envelope. Alternatively, two individual PN could be formed first but quickly undergo membrane fusion resulting in a so-called ‘syngamy nucleus’. In contrast, Azevedo et al. (2014) proposed premature pronuclear envelope breakdown (pPNBD) as another mechanism in single pronucleated zygotes. Because of a low rate of normal diploidy in affected zygotes, the latter authors do not recommend to use the resulting embryos for transfer. These brief considerations show that continuous monitoring of many oocytes during the period between sperm penetration and appearance of PN is necessary to obtain more information on such phenomena, for instance to ascertain whether early pronuclear membrane fusion exists and to assess the frequency of pPNBD. In short, time-lapse monitoring represents a promising technique that hopefully will be developed further to satisfy all specific laboratory requirements and that may help to address some yet unresolved scientific issues.
Cytogenetic analysis of a presumably tripronuclear zygote revealed that triploidy was caused by an endoreduplicated 46,XX complement. The remaining chromosomes yielded a hyperhaploid karyotype of 28,Y, +2, +3C, +D. The origin of this chromosomal composition is obscure. Besides endoreduplication in a normal 23,X oocyte pronucleus and fertilization by a normal 23,Y spermatozoon, an additional female pronucleus might have been formed due to an irregular chromosome distribution during second meiotic division. On the other hand, penetration by a hyperhaploid spermatozoon cannot be excluded with certainty.
A-A.Baschat and KJXedrich partner will need to be screened for heterozygosity of the cystic fibrosis gene.It would seem that performing ICSI using spermatozoa from chromosomally normal males does not increase the risk of fetal chromosomal abnormalities.Therefore we do not recommend prenatal diagnosis on the basis of ICSI alone unless the woman carries an independently increased risk of chromosomal aberration in the offspring.However, in our opinion, the high incidence of abnormal karyotypes in the male subfertile population clearly warrants genetic testing of the male partner prior to ICSI.If pathology is discovered prenatal diagnosis should be performed. References
Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta- adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone.
Simultaneously with in vitro fertilization (IVF), the acridine orange (AO) test was performed on 51 patients according to the method of Tejada et al. This test allows a differentiation of spermatozoa with intact (double-stranded) and denatured (single-sanded) DNA. A significant relationship was demonstrated between the percentage of green-fluorescing (intact) sperm and the results of IVF. For both, the groups with (n = 40) and without (n = 11) fertilization of human eggs, a positive correlation was found between AO-test and sperm motility, and AO-test and normal sperm morphology.
