To What Extent Are Cryopreserved Sperm and Testicular Biopsy Samples Used in Assisted Reproduction?

BACKGROUND: Testicular biopsies and ejaculated spermatozoa are routinely cryopreserved in many units but the fate of these samples has not provoked large interest. This prompted us to review our data accumulated during a period of 20 years (1997 to 2016). METHODS: For patients with biopsies (group 1) or ejaculated spermatozoa (group 2), an attempt was made to evaluate whether the samples stored, had been discarded with the patient's consent or because the patient had died, or whether they had been transported to another laboratory. In each of these categories, a previous use in our program of assisted reproduction was assessed. RESULTS: The total utilization rate in group 1 (n=95) was 53.7% and only 5.48% in group 2 (n=365). In both groups, deceased patients had not previously used their cryopreserved samples. In detail, the utilization rates for still banked, discarded and transferred samples were 84.2%, 50% and 27.3%, respectively in group 1 and 2.88%, 10.4% and 10%, respectively in group 2. CONCLUSION: The exact reasons for the low utilization rates of cryopreserved male gametes remain to be explored. A closer contact between sperm banking units and patients might be useful to discuss the need for further storage of the probes, their possible disposal or the prospects when a specific use for assisted reproduction is intended.

The chromosomal constitution of embryos arising from monopronuclear oocytes in programmes of assisted reproduction.

The assessment of oocytes showing only one pronucleus during assisted reproduction is associated with uncertainty. A compilation of data on the genetic constitution of different developmental stages shows that affected oocytes are able to develop into haploid, diploid, and mosaic embryos with more or less complex chromosomal compositions. In the majority of cases (~80%), haploidy appears to be caused by gynogenesis, whereas parthenogenesis or androgenesis is less common. Most of the diploid embryos result from a fertilization event involving asynchronous formation of the two pronuclei or pronuclear fusion at a very early stage. Uniparental diploidy may sometimes occur if one pronucleus fails to develop and the other pronucleus already contains a diploid genome or alternatively a haploid genome undergoes endoreduplication. In general, the chance of obtaining a biparental diploid embryo appears higher after conventional in vitro fertilization than after intracytoplasmic sperm injection. If a transfer of embryos obtained from monopronuclear oocytes is envisaged, it should be tried to culture them up to the blastocyst since most haploid embryos are not able to reach this stage. Comprehensive counselling of patients on potential risks is advisable before transfer and a preimplantation genetic diagnosis could be offered if available.

The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities.

Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism, chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the formation of multiovular follicles.

A preliminary concept, deduced from cytogenetic analyses, for explaining different types of multipronuclear oocytes obtained after intracytoplasmic sperm injection.

Oocytes with three or more pronuclei and one or two polar bodies arising after intracytoplasmic sperm injection were analyzed cytogenetically, and the data were used to develop a general model explaining the possible origin of the abnormal pronuclear stages. The mechanisms, either separately or in variable combinations, that induce the appearance of additional pronuclei are: 1) nonextrusion of the second polar body; 2) incomplete chromatid segregation into the extruded second polar body; and 3) dispersal of the oocyte chromatids in the presence of the second polar body formation.

Separation of a pronucleus by premature cytokinesis: a mechanism for immediate diploidization of tripronuclear oocytes?

OBJECTIVE: To describe an oocyte with a peculiar combination of abnormalities in terms of cytoplasmic fragmentation and formation of pronuclei. DESIGN: Case report. SETTING: University-based IVF unit. PATIENT(S): A chromosomally normal couple undergoing the third treatment by assisted reproduction. INTERVENTION(S): Controlled ovarian hyperstimulation, follicular aspiration, testicular sperm extraction, intracytoplasmic sperm injection (ICSI), and oocyte fixation. MAIN OUTCOME MEASURE(S): Assessment of pronuclear formation and cytogenetic analysis of a tripronuclear fragmented oocyte. RESULT(S): The described oocyte revealed two polar body-like structures after follicular aspiration and developed three pronuclei after ICSI, accompanied by extrusion of another polar body-like globule. Moreover, the pronuclear stage had undergone a premature irregular cytokinesis, including one of the pronuclei in the smaller half of the divided cytoplasm. Cytogenetic analysis showed a triploid karyotype (23,X/23,X,ace/23,Y) commonly found in digynic ICSI zygotes. CONCLUSION(S): The chromosomal constitution suggests that the third globule represents another cytoplasmic fragment and no regular second polar body. Separation of a supernumerary pronucleus by premature cytokinesis might be relevant for the discussion on diploidization of triploid zygotes.

The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center.

PURPOSE: To investigate the effects of male aging on sperm quality and sperm DNA fragmentation. METHODS: The ejaculates of 320 unselected men attending a fertility clinic and, as a control, 84 normozoospermic men without any history of ART were analyzed according to WHO guidelines. Sperm DNA fragmentation was measured by flow cytometry after staining with propidiumiodide. RESULTS: The patients were divided into four groups: <30 years, 30-35 years, 36-39 years and >or=40 years. Sperm motility decreased with increasing age whereas sperm concentration, morphology, and DNA fragmentation fluctuated throughout the four groups both among patients and among controls. However, we could not detect any significant correlation between male age and conventional semen parameters or sperm DNA fragmentation, respectively, neither in the patients' group nor among the controls. This also applies to a classification of patients and controls into only two age groups with a cut-off point at 35 years. CONCLUSIONS: Our findings suggest that neither the routinely assessed semen parameters nor the amount of spermatozoa with fragmented DNA are affected by male age.

Selective microsurgical removal of a pronucleus from tripronuclear human oocytes to restore diploidy: disregarded but valuable?

OBJECTIVE: To review data on the microsurgical removal of a single pronucleus from tripronuclear human oocytes and evaluate the future potential of this technique for obtaining diploid, transferable embryos. DESIGN: Literature review. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULT(S): Ten relevant studies were identified. These differ considering the removal technique itself, the application of cytoskeletal relaxants, and the survival rate after epronucleation. Diploidy and heteroparental inheritances could be confirmed in some preimplantation stages derived from epronucleated oocytes. Transfer of "corrected" embryos has been attempted only once, and resulted in a live birth. Noteworthy pitfalls associated with the procedure concern the exact identification of the supernumerary pronucleus, the presence of two centrosomes in dispermic oocytes, and cytogenetically abnormal pronuclear patterns after intracytoplasmic sperm injection. CONCLUSION(S): Patients with exclusively abnormally or few normally fertilized oocytes would profit from epronucleation to assure embryo transfer or increase the number of transferable embryos. Further research appears necessary and promising.

Human oocyte chromosome analysis: complicated cases and major pitfalls.

Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes.

Mechanisms giving rise to triploid zygotes during assisted reproduction.

OBJECTIVE: To review information on the origin of triploid zygotes as gathered from assisted reproduction techniques. DESIGN: Identification of relevant literature by a MEDLINE search and own experience on the basis of cytogenetic studies of abnormally fertilized oocytes. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULT(S): Penetration of two haploid spermatozoa or of a single diploid spermatozoon into the oocyte causes diandric triploidy. The first case can be discerned by formation of a total of three pronuclei, whereas the second process will remain undetected, because it involves a female and a single but diploid male pronucleus. Digynic triploidy after intracytoplasmic sperm injection is characterized by nonextrusion of the second polar body and formation of three pronuclei. Digyny can also result from the fertilization of diploid giant oocytes. Depending on how maturation of these gametes proceeds, three or only two pronuclei will be observed. Thus, the size of the pronuclear stage must be considered for a successful identification of the abnormality. Endoreduplication within the female pronucleus is not detectable and may represent another, albeit rare, origin of digynic triploidy. CONCLUSION(S): Routine inspection of the number of pronuclei is not an absolutely reliable tool for excluding the development of triploid embryos. Observations during assisted reproduction may yield valuable information on the origin of triploidy.

Cytogenetic analysis of human oocytes remaining unfertilized after intracytoplasmic sperm injection.

Oocytes remaining unfertilized after intracytoplasmic sperm injection showed 12.0% aneuploidy (nondisjunction + unbalanced predivision), 3.4% structural aberrations, and 8.5% balanced predivision in fully karyotyped cells. However, the frequently observed complete or partial separation of chromatids, most probably caused by abortive activation, might complicate the evaluation of meiosis I-derived aneuploidy and questions the relevance of balanced predivision.

The incidence of aneuploidy in human oocytes assessed by conventional cytogenetic analysis.

Human oocytes failing to fertilize during assisted reproduction are an important source of information for assessing incidence and causal mechanisms of maternal aneuploidy. This review describes the techniques of conventional oocyte chromosome analysis and evaluates the results of 59 studies comprising a total of>10,000 female gametes. The mean rate of aneuploidy (hypohaploidy + hyperhaploidy) amounts to approximately 20%, but this incidence is reduced as soon as possible artifacts introduced by the fixation technique are taken into consideration. It is therefore concluded that a realistic value for numerical abnormalities arising during first meiotic division lies between 12 and 15%. All chromosome groups are affected by aneuploidy but the actually observed frequencies exceed the expected frequencies in groups D, E, and G. Two aneuploidy-causing mechanisms have been identified in human oocytes: nondisjunction, resulting in the loss or gain of whole chromosomes, and predivision, resulting in the loss or gain of single chromatids. A brief analysis including only aneuploid complements with one extra or missing chromosome/chromatid shows a slight increase in predivision (52.9%) compared with nondisjunction (47.1%). Finally, suggestions for future studies are given since, for instance, the presentation of results and the use of cytogenetic nomenclature have not been uniform.

Endoreduplication of the hyperhaploid maternal complement and abnormal pronuclear formation in a human zygote obtained after intracytoplasmic sperm injection.

We report the cytogenetic analysis of a tripronuclear zygote with two polar bodies observed after intracytoplasmic sperm injection. Rare previous investigations of this kind of zygote suggested a diploid or a hypotriploid chromosome constitution. In contrast, the present case turned out to be hypertriploid. Besides the haploid (23,Y) sperm chromosome set, there was a hyperdiploid endoreduplicated (end48,XX,+18,+18) maternal contribution. This zygote not only revealed a peculiar combination of different anomalies (hyperhaploidy of the female gamete, endoreduplication and abnormal pronuclear formation) but also indicates that endoreduplication may sporadically contribute to the generation of triploidy.