The (t)WE lab Simplified IVF Procedure: First Births after freezing/thawing.

BACKGROUND: Infertility care is one of the most neglected health care issues in developing countries (DC), affecting more than 50 million couples. The social stigma of childlessness still leads to isolation and abandonment. Bilateral tubal occlusion due to sexually transmitted diseases and pregnancy-related infections is the most common cause of infertility in DC. Consequently most cases of infertility are only treatable by using assisted reproductive technologies which are either unavailable or too costly. Lowering the laboratory costs associated with IVF is a crucial step to make IVF affordable for a larger part of the world population. We recently developed and described a new simplified method of IVF culturing, called the (t)WE lab method. Our initial results in fresh IVF cycles showed that IVF methodology can be significantly simplified and result in successful outcomes at levels that compare favourably to those obtained in high resource programs. CASE-REPORTS: We report three pregnancies and four live births as a result of transferring five cryo/thawing embryos which were developed after using the simplified (t)WE lab system. The two singleton babies delivered vaginally, for the twin pregnancy a caesarean section was performed. All babies were healthy, the perinatal outcome was uneventful in all cases. CONCLUSION: We provide proof-of-principle evidence that transferring cryopreserved/thawed embryos obtained with our (t)WE lab simplified culture system can lead to successful pregnancies and healthy live births.

A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos.

The occurrence of a pleiomorphic population of cytoplasmic fragments is a common characteristic of early human embryos fertilized in vitro. Here, temporal, spatial, fine structural, and biochemical aspects of fragmentation were examined in fragmented monospermic and dispermic pronuclear to early cleavage stages human embryos classified as stage-appropriate during the first 3.5 days of culture. The morphodynamics of certain common patterns of fragmentation and the movement and composition of fragments were analysed by time-lapse video, mitochondrial fluorescent probes, and transmission electron microscopy. Plasma membrane and nuclear DNA integrity were assessed by annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and single-cell alkaline gel electrophoresis ('comet') assays respectively. Developmental competence for affected embryos was related to outcome after embryo transfer. The results demonstrate that certain common forms of spontaneous fragmentation affecting early human embryos are not lethal, and that clusters of apparent fragments are often transient structures, which disappear by resorption or lysis. The findings suggest that the occurrence and fate of fragments characteristic of these phenotypes may be related to oncosis-like processes associated with transient and focal ATP deficiencies in blastomeres and mitochondrial deficiencies or absence in extracellular fragments.

Differential effects of repeated ovarian stimulation on cytoplasmic and spindle organization in metaphase II mouse oocytes matured in vivo and in vitro.

The effects of four rounds of ovarian stimulation spaced 1-6 weeks apart on the normality of metaphase II (MII) spindle formation, chromosomal alignment and cytoplasmic organization were examined in intact ovulated mouse oocytes and at MII for oocytes obtained at the germinal vesicle stage from the same ovaries and matured in vitro. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay was used to identify DNA strand breaks in chromosomes, and histological studies of ovaries between and at each round of ovarian stimulation were performed. The results demonstrate a progressive and significant increase in the frequency of spindle defects with each round of ovarian stimulation, including those spaced weeks apart. Oocytes with spindle defects were also characterized by the occurrence of detached chromosomes and cytoplasmic asters. In contrast, in-vitro matured oocytes derived from the same ovaries were normal. No evidence of DNA strand breaks with repeated rounds of ovarian stimulation was detected in ovulated or in-vitro matured oocytes. The development and persistence of nodules of hypertrophied granulosa in regions where follicular growth occurs suggest that a progressively increasing proportion of oocytes in the ovulatory pathway may experience an intrafollicular milieu that has negative consequences for competence. The results are discussed with respect to ovarian and oocyte biological ageing and possible adverse implications for human oocyte competence with repeated hyperstimulation.

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence.

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.

The vascular character of ovarian follicular granulosa cells: phenotypic and functional evidence for an endothelial-like cell population.

Ovarian follicular granulosa cells express temporally and spatially distinct functions throughout the follicle cycle. During the entire cycle, granulosa cells exhibit an unusually broad range of activities including the secretion of steroid hormones, enzymes, growth factors and cytokines. To date, the identity(ies) of these cells (lineage/cell type) remains unknown. We demonstrate expression of the Tie, Tek, cKit, Flt-1, CD-31 and vWF proteins and the ability to rapidly internalize acetylated low density lipoprotein among mural and cumulus subpopulations of human and murine follicular granulosa cells. In addition, we provide evidence that human and murine granulosa cells can engage in tube-forming activity in vitro. To the best of our knowledge, the six phenotypic and two functional markers examined during this study, as a group, are associated only with endothelial or endothelial-like cells. In total, the findings suggest that some granulosa cells may have the potential to actively participate in the vascularization of the corpus luteum, by way of an inherent capacity which is likely to be a characteristic of their unique identity and lineage. This inherent capacity of granulosa cells to behave and respond, at least to some extent, like endothelial cells may be of possible importance in the aetiology of certain follicular pathologies.

Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function.

While genetic and epigenetic factors have been associated with the developmental competence of human oocytes and embryos produced by IVF, how such factors develop or influence the oocyte remain to be explained. This paper reviews evidence which suggests that the degree of perifollicular vascular expansion associated with increased rates of blood flow are developmentally important for the generation of a normal follicle and competent oocyte. The degree of vascular development is follicle specific and differences between follicles might reflect their unique abilities to regulate angiogenic growth factor(s) production by the follicle cells in response to hypoxia. The notion that mitochondrial function in oocytes and early embryos could be influenced by intrafollicular conditions, and that differential patterns of mitochondrial segregation occur in blastomeres during early cleavage, is discussed with respect to the role of these organelles as critical determinants of developmental competence.

Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains.

This study examined the relationship between blastomere fragmentation in cultured human embryos obtained by in-vitro fertilization and the effect of fragmentation on the distribution of the following eight regulatory proteins found to be: (i) localized in the mature oocyte in subplasmalemmal, polarized domains; and (ii) unequally inherited by the blastomeres during cleavage: leptin, signal transducer and activator of transcription 3 (STAT3), Bax, Bcl-x, transforming growth factor beta 2 (TGF beta 2), vascular endothelial growth factor (VEGF), c-kit and epidermal growth factor R (EGF-R). Four basic patterns of fragmentation were observed. The severity of the impact of each type of fragmentation on the affected blastomere(s) and the developmental competence of the embryo appeared to be a function of the unique temporal and spatial features associated with the particular fragmentation pattern(s) involved in each instance. The findings demonstrate that certain patterns of fragmentation can result in the partial or near total loss of the eight regulatory proteins from specific blastomeres and that the developmental potential of the affected embryo can be particularly compromised if it occurs during the 1- or 2-cell stages. In contrast, fragmentation from portions of a fertilized egg or a blastomere(s) in a 2-cell embryo that do not contain the protein domains, or the complete loss by fragmentation of a regulatory protein domain-containing blastomere after the 4-cell stage does not necessarily preclude continued development to the blastocyst, although the normality and developmental potential of the embryo may be compromised. The possible association between fragmentation and apoptosis was examined by annexin V staining of plasma membrane phosphatidylserine and TUNEL analysis of blastomere DNA. No direct correlation between fragmentation and apoptosis was found following the analyses of fragmented embryos with these two markers. However, while we suggest that changes in cell physiology unrelated to apoptosis are the more likely causes of fragmentation, we cannot exclude the possibility that fragmentation itself may be an initiator of apoptosis if critical ratios or levels of developmentally important proteins are altered by partial or complete elimination of their polarized domains. The findings are discussed with respect to the possible developmental significance of regulatory protein polarization in human oocytes and preimplantation stage embryos.

Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy.

The developmental competence of mouse and human early embryos appears to be directly related to the metabolic capacity of a finite complement of maternally inherited mitochondria that appear to begin to replicate after implantation. Mitochondrial dysfunctions resulting from a variety of intrinsic and extrinsic influences, including genetic abnormalities, hypoxia and oxidative stress, can profoundly influence the level of ATP generation in oocytes and early embryos, which in turn may result in aberrant chromosomal segregation or developmental arrest. Deletions and mutations in oocyte mitochondrial DNA may subtend metabolic deficiencies or replication disorders in some infertile women and in women of increased reproductive age. Here, we describe methods for (i) the compartmentalization of mouse and human oocyte mitochondria into unique cytoplasts enriched for these organelles, and (ii) their transfer by microinjection into intact recipient oocytes. Metabolically active mitochondria in donor and recipient metaphase II stage oocytes were labelled with mitochondria-specific fluorescent probes, and the fate and location of donated mitochondria in recipient oocytes were followed by conventional epifluorescence and scanning laser confocal fluorescence microscopy. The net ATP content of undisturbed and recipient oocytes from the same cohort(s) was measured quantitatively at timed intervals after mitochondrial injection. The results demonstrate the feasibility of isolating and transferring mitochondria between oocytes, an apparent increase in net ATP production in the recipients, and the persistence of activity in the transferred mitochondria. The findings are discussed with respect to mitochondrial function and dysfunction in mammalian oocytes and embryos, and to the potential clinical applications of mitochondrial donation as they relate to the creation of heteroplasmic embryos.

DNA strand breaks and phosphatidylserine redistribution in newly ovulated and cultured mouse and human oocytes: occurrence and relationship to apoptosis.

This study determined the occurrence of two molecular markers of apoptosis, chromosomal DNA strand breaks and oolemma phosphatidylserine redistribution, in >200 uninseminated and unfertilized human oocytes, and >800 newly ovulated and cultured mouse oocytes. DNA breaks were analysed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and phosphatidylserine by annexin V staining, with imaging by conventional epifluorescence and scanning laser confocal fluorescence microscopy. More than 300 intact and 500 fragmented mouse oocytes were examined at 24 h intervals during 6 days of culture in three different types of medium. For the human, 205 oocytes were examined at retrieval or at 24 h intervals during 7.5 days of culture in two types of medium. The perifollicular vascularity and the dissolved oxygen content of follicular fluid were determined for most of the follicles from which human oocytes were derived. The results demonstrate that TUNEL fluorescence of metaphase II (MII) chromosomes and annexin V staining of the oolemma in newly ovulated and cultured mouse and human oocytes are rare, and, when detected, are not spatially or temporally related. This finding also applied to mouse oocytes that fragmented during culture and exhibited morphological features that grossly resembled apoptotic body formation. In contrast, TUNEL but not annexin V staining occurred in the first polar body of a relatively high proportion of newly ovulated mouse oocytes, but was rarely detected in newly aspirated human oocytes. For the human, the occurrence of MII chromosomal TUNEL fluorescence was patient-specific and unrelated to perifollicular vascularity or dissolved oxygen content of the corresponding follicular fluid. The pattern of chromosomal TUNEL fluorescence observed in the first polar body and in the MII chromosomes of a very small number of mouse and human oocytes, especially after many days of culture, suggests that DNA strand breaks may not arise by apoptosis-associated endonuclease digestion. The results with these two markers suggest that it is premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects.

Epigenetic influences on oocyte developmental competence: perifollicular vascularity and intrafollicular oxygen.

PURPOSE: Studies indicating that the developmental competence of the human oocyte is influenced by the level of intrafollicular oxygen are described. METHODS: Perifollicular vascularity and dissolved oxygen content were determined by color Doppler ultrasonography and analysis of follicular fluid at ovum retrieval, respectively, in stimulated cycles for in vitro fertilization. RESULTS: Differences in the degree of perifollicular vascularity correlate with differences in the dissolved oxygen content of the corresponding follicular fluid. Oocytes with cytoplasmic and chromosomal disorders and embryos with multinucleated blastomeres and limited developmental ability were derived predominantly from underoxygenated follicles. Findings from several studies indicate that embryos with the highest implantation potential originate from follicles that are well-vascularized and oxygenated. CONCLUSIONS: Follicular vascularity and oxygen content appear to be important determinants of oocyte competence. Possible causes of differences in follicle-specific vascularity and the potential effects of severe hypoxia on the normality of molecular and cellular processes during follicle growth and preovulatory development are discussed.

A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor-beta2 sequestration in a subpopulation of human ovarian follicle cells.

This study describes the occurrence of a highly specialized subpopulation of granulosa and cumulus oophorus cells that accumulate and sequester specific growth factors by a novel mechanism. These cells are characterized by multiple balloon-like processes tethered to the cell by means of a slender stalk of plasma membrane. Time-lapse analyses demonstrate that these tethered structures (TS) form in minutes and frequently detach from the cell with the bulbous portion remaining motile on the cell surface. Serial section reconstruction of transmission electron microscopic images shows a specific and stable intracellular organization in which an apparent secretory compartment composed of densely packed vacuoles, vesicles, and cisternae is separated by a thick filamentous network from a nuclear compartment containing mitochondria, polyribosomes, lipid inclusions, and rough-surfaced endoplasmic reticulum. Immunofluorescent analysis performed during the formation of these structures showed a progressive accumulation of vascular endothelial growth factor, leptin, and transforming growth factor-beta2 in the bulbous region. TS were identified in newly aspirated masses of granulosa and cumulus oophorus, and their production persists for months in culture. Observations of TS-forming cells made over several days of culture indicates that their production is episodic and factor release from these cells may be pulsatile. The findings suggest that a novel method of growth factor storage and release by an apparent apocrine-like mechanism occurs in the human ovarian follicle. The results are discussed with respect to possible roles in pre- and post-ovulatory follicular development.

The expression of leptin and its receptors in pre-ovulatory human follicles.

The expression of leptin and its receptors was examined by reverse transcriptase-polymerase chain reaction and immunofluorescence in granulosa and cumulus cells of pre-ovulatory follicles and in meiotically mature oocytes obtained from women undergoing in-vitro fertilization. Leptin concentrations were measured in newly aspirated follicular fluids and in maternal serum before and after the administration of an ovulatory dose of human chorionic gonadotrophin. The findings demonstrate leptin expression at the mRNA and protein levels by granulosa and cumulus cells, and the presence of leptin in mature human oocytes. While an association between follicular leptin concentration and embryo development was not observed, a post-ovulatory increase in serum leptin concentration was associated with implantation potential. The results are discussed with respect to possible roles of leptin in early human development.

The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics.

Regardless of whether fertilization occurs in vivo or in vitro, a large proportion of human embryos do not develop progressively through the pre-implantation stages or arrest development after implantation. This study examined the association between the chromosomal/spindle normality of the mature human oocyte and the dissolved oxygen content, vascular endothelial growth factor concentration (VEGF) and perifollicular blood flow characteristics of the corresponding ovarian follicles. Findings from >1000 samples of follicular fluid show that developmentally significant differences in dissolved oxygen content occur in follicular fluids aspirated from follicles of equivalent size and ultrasonographic appearance. Oocytes from severely hypoxic follicles were associated with high frequencies of abnormalities in the organization of the chromosomes on the metaphase spindle that could lead to segregation disorders and catastrophic mosaicisms in the early embryo. Oocytes with cytoplasmic defects and cleavage stage embryos with multinucleated blastomeres are derived predominantly from severely hypoxic follicles. VEGF measurements of follicular fluid and colour pulsed Doppler ultrasonographic analysis of follicle-specific blood flow characteristics indicated a potentially important role for this factor both in perifollicular angiogenesis and in the regulation of intrafollicular oxygen levels. The results are discussed with respect to how severe intrafollicular hypoxia may influence the normality of chromosomal organization and segregation in the oocyte, and whether detailed pulsed Doppler analysis of individual pre-ovulatory follicles may provide an indirect indication of the 'health' of the follicle and possibly the developmental competence of the corresponding oocyte.

Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo.

Unique protein domains, concentration gradients, and asymmetric protein distributions or polarities are principle forces establishing the identity and fate of individual cells during early development in lower vertebrates and invertebrates. Here, we present evidence that these same forces exist during mammalian development in the form of two representative regulatory proteins, leptin and STAT3. Leptin, the 16 kDa cytokine product of the obese gene (ob) is involved in the activation of STAT3, a member of the signal transducer and activation of transcription family of proteins. We examined the temporal and spatial aspects of leptin and STAT3 immunofluorescence in mouse and human oocytes and preimplantation stage embryos. The findings demonstrate that both leptin and STAT3 are polarized in the oocyte and, as a consequence of their location and the position of the cleavage planes with respect to these protein domains: (i) differences in allocation of these proteins between blastomeres occur at the first cell division such that by the 8-cell stage; (ii) unique cellular domains consisting of leptin/STAT3 rich and leptin/STAT3 poor populations of cells are generated. By the morula stage, a cell-borne concentration gradient of these proteins extending along the surface of the embryo is observed. A potential role of these proteins in early development is indicated at the morula stage where the 'inner' cells consist of blastomeres that contain little, if any, leptin/STAT3 while 'outer' cells contain both leptin/STAT3 rich and poor cells. This pattern persists through the hatched blastocyst stage with little, if any, leptin/STAT3 detected in the inner cell mass and populations of leptin/STAT3 rich and poor cells forming the trophoblast. We have examined oocytes from mutant C57BL/6J ob/ob mice which are both obese and infertile (although fertility can be restored by the exogenous provision of leptin) and have found STAT3 and the mutant (truncated) leptin protein to be present and polarized, suggesting the possibility that the truncated leptin protein may still contain operational domains which are functional during oocyte development and early embryogenesis. Furthermore, analysis of leptin and STAT3 in intact ovarian follicles suggests that these proteins may be maternally derived and in particular, that a subpopulation of follicle cells may be partly responsible for the establishment of their polarized distribution in the oocyte. The results are discussed with respect to the proposition that leptin and STAT3 have critical roles in early mammalian development, and may be involved in the determination of the animal pole of the oocyte and in the establishment of the inner cell mass and trophoblast in the preimplantation stage embryo.

The oxidizing agent tertiary butyl hydroperoxide induces disturbances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes.

It has been recently proposed that a concomitant generation of oxidative stress of oocytes with increasing maternal age may be a major factor responsible for the age-related increase in aneuploid conceptions. As a preliminary step in the testing of this hypothesis, we need to confirm that oxidative stress in itself can induce errors in chromosome segregation. In order to achieve this goal, germinal vesicle (GV)-stage mouse oocytes from unstimulated ICR and (C57BL x CBA) F1 hybrid female mice were matured in vitro for 9 h for metaphase I (MI) oocytes or 16 h for metaphase II (MII) oocytes in the presence of varying concentrations of the oxidizing agent tertiary-butyl hydroperoxide (tBH). MII oocytes from (C57BL x CBA) F1 hybrid mice were fixed and C-banded for karyotyping analysis. MI and MII oocytes from ICR mice were fixed and stained with the DNA-fluorescent probe 4',6-diamidino-2-phenylindole (DAPI) to detect abnormalities in chromosomal distribution. Meiosis I and meiosis II spindles from ICR mice were visualized by confocal immunofluorescence microscopy. Data from these experiments demonstrate that in-vitro exposure of mouse oocytes to tBH during meiosis I reduces the length (pole-to-pole distance) and width (diameter at the equator of the spindle) of meiosis I and meiosis II spindles. This reduction is associated with an increase in the percentage of oocytes showing chromosome scattering and clumping on the MII plate, and of aneuploidy (hyperhaploidy) in MII oocytes. However, tBH at the concentrations used in the present study has only a minimal negative effect on the frequency of meiotic maturation. These results suggest that oxidative stress during meiotic maturation in vitro may induce chromosomal errors that are undetectable in the living oocyte and whose developmental consequences may become manifest after fertilization.

Sperm centrosome dysfunction: a possible new class of male factor infertility in the human.

This study examined the normality of microtubule growth and aster development from the sperm centrosome in failed fertilizations and pronuclear-arrested eggs after intracytoplasmic insemination with spermatozoa derived from severe oligoteratozoospermic individuals. In these cases, male factor was of unknown origin or was associated with vasectomy reversal in men with known prior fertility. The findings indicate that an impaired capacity to promote the growth of microtubule arrays from the sperm centrosome may preclude pronuclear opposition required for syngamy. Centrosome dysfunction may contribute to the developmental arrest of human embryos at the pronucleate stage and consequently may represent a new class of sperm defects associated with early human developmental failure.

The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte.

The relationship between factors external to the human oocyte and the developmental potential of the female gamete after meiotic maturation in vivo is discussed. The findings indicate very different intrafollicular oxygen contents exist among follicles from the same and different patients, and marginally hypoxic conditions in some follicles are associated with a comparatively low adenosine triphosphate content and a reduction in intracellular pH for the corresponding metaphase II-stage oocyte. For such oocytes, the finding of an increased frequency of cytoplasmic pathology and chromosomal scattering (aneuploidy) suggests a compromised developmental potential. Three distinctly different patterns of cumulus cell attachment and proliferation during the first 24 hours of culture after insemination are described. As reported earlier, different cumulus cell phenotypes observed in vitro are independent of the presence of the oocyte or fertilized egg and unrelated to the degree of expansion of the cumulus at retrieval. However, developmental viability and implantation potential after in vitro fertilization-embryo transfer appear to be related to an intrinsic pattern of cumulus cell behavior in vitro. The results are discussed with respect to the role of factors external to the human oocyte that may influence or determine the ability of the oocyte to develop progressively after fertilization.

Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human.

This report describes spatial and temporal aspects of sperm penetration and intracytoplasmic migration, pronuclear evolution and the specificity of presyngamic opposition, stage-specific changes in cytoskeletal organization and the relative contribution of maternal and paternal components to mitotic spindle formation. These studies involved observations of living human oocytes during conventional insemination in vitro and after intracytoplasmic deposition of spermatozoa, analysis of chromatin organization and distribution during pronuclear evolution, and detection of actin and alpha-, beta- and gamma-tubulin by confocal immunofluorescence microscopy. Immature and mature oocytes, penetrated but unfertilized oocytes, fertilized but arrested eggs, and cleavage-stage embryos from normal and dispermic fertilizations were examined. The results demonstrate that sperm nuclear migration to the maternal perinuclear region is rapid and linear, occurs in the absence of a detectable cytoskeletal system and appears to be assisted by an unusual configuration of the sperm tail principal piece which results from either retained intracytoplasmic motility or the process by which the sperm tail is progressively incorporated into the oocyte. Our findings also show a specificity of pronuclear alignment that is associated with a polarized distribution of both maternal and paternal chromatin, and with the position of the sperm centrosome and the presence of microtubules nucleated from this structure. The results also indicate that a maternal microtubule nucleating capacity is present in the immature oocyte but is apparently inactive until spindle formation. The poles of the first mitotic spindle appear to be derived from the sperm centrosome, although some maternal contribution cannot be excluded. The sperm tail and centrosome persist in a single cell through the cleavage stages, and the latter serves as a prominent site of cytoplasmic microtubule nucleation. The results provide a detailed understanding of the cellular and nuclear morphodynamics of the human fertilization process and indicate subtle defects that may be responsible for early developmental failure.

Evolution of the sperm aster after microinjection of isolated human sperm centrosomes into meiotically mature human oocytes.

This study examined the feasibility of isolating and transferring the centrosome-containing region of spermatozoa to mature human oocytes. The findings demonstrate that individual sperm centrosomes can be transferred and are capable of nucleating maternal tubulin to form a well-developed sperm aster in the recipient oocyte. The results are discussed with respect to centrosome function in early human development and applications in clinical in-vitro fertilization in the treatment of certain forms of male factor infertility.