INTRODUCTION

Before fertilisation, the mammalian oocyte is arrested at metaphase of the second meiotic division (MII), and it only resumes meiosis upon penetration by a spermatozoon. Approximately 16 to 18 hours after fertilisation, two pronuclei (2PN) form; one is derived from the oocyte and encloses the maternal genome, whereas the other originates from the spermatozoon and contributes the paternal complement (2). The pronuclear stage is followed by the fusion of both pronuclei in a process called syngamy, which results in the diploid zygote (3, 4) (Figure 1). However, dysregulation of any of the processes involved may cause unusual cytogenetic events, and it may occasionally be associated with an abnormal pronuclei number (5). Triploidy is characterised by the presence of an additional chromosome complement, often recognised by the presence of three pronuclei (3PN) (2). 3PN is the most common chromosomal abnormality to impact human gestation, accounting for 17% of all spontaneous miscarriages (4, 6-8). Triploidy is an embryonic lethal event and, as such, it prevents normal embryonic development in most cases. Yet, several live births have been reported (9, 10).

A recent case report of sex-discordant monochorionic twins has challenged the notion that monochorionic twins are definitely monozygotic (1) (see our previous post). This phenomenon is explained by a postulated third type of twinning referred to as sesquizygosity, where individuals share between 50% to 100% of their genetic identity. Unlike monozygotic and dizygotic cases, the underlying mechanisms of this rare event are less extensively defined (1, 11). However, since the advent of advances in ART, the mechanisms involved in triploidy can be further elucidated to provide an explanation for sesquizygosity.

The origin of triploidy may be classified as one of two occurrences: diandry or digyny, resulting in an extra paternal or maternal haploid chromosome set, respectively. Each event comprises numerous postulated mechanisms involving the control of sperm entry into the oocyte, pronuclei formation, mitotic spindle formation and initiation of meiosis.

The proposed mechanisms of triploid embryo formation and mechanisms explaining sesquizygotic twinning are discussed below, considering the relevance and incidence of triploid embryos in IVF.

Process normal fertilisation mammals

Figure 1 . Process of normal fertilisation in mammals (12).

TRIPLOID INCIDENCE AND RELEVANCE TO ART

In the context of ART, 5% to 7% of all IVF or ICSI cycles result in at least one abnormal pronuclei event (4, 13, 14). In particular, triploidy has been demonstrated to occur at a slightly lower, yet significant rate in ICSI cycles (2.5%) when compared to IVF (8.1%) (3, 15). Such a difference is presumably explained by the fact that ICSI reduces the probability of polyspermic fertilisation (3, 15).

Multiple studies have associated 3PN with low blastocyst formation and implantation rates, which provides a rationale for routine pronuclei observation in ART procedures (16). This clinical laboratory practice serves as a prognostic indicator of embryo competence and normal fertilisation (5, 6, 16). Normally, zygotes with variation from 2PN and two polar bodies are discarded in order to increase the chances of selecting the best embryo for transfer (3, 15). However, it is important to note that visualisation of 2PN at the pronuclear stage does not always eliminate the possibility of triploidy, and triploid embryos may cleave at a rate comparable to normally fertilised embryos (Figure 2) (6, 8, 15). Additionally, while IVF allows for the examination of the pronuclear stage, the incidence of 3PN is presumed to occur in vivo at a similar rate; however, these embryos are usually unable to pass developmental checkpoints and result in miscarriage (17).

Although the factors and mechanisms contributing to triploidy remain controversial, the incidence of 3PN in assisted reproduction cycles has been associated with advanced maternal age or semen abnormalities in some cases (18). Additionally, a link between 3PN and maternal genetic cause, high gonadotropin dose and large oocyte yields during ovarian stimulation has also been established (19, 20). Therefore, the complex relationship between ART and the incidence of triploidy is likely to be of multifactorial aetiology.

Development of 2PN and 3PN human embryos. (A) 2PN fertilised oocyte. (B) Same 2PN zygote during the  first cleavage. (C) Diploid embryo on day 3 after fertilisation. (D) 3PN fertilised oocyte. (E) Same 3PN zygote during first cleavage with a tripolar spindle. (F) Triploid embryo on day 3 after fertilisation. Note similar morphological appearance on day 3 compared to 2PN in C (17).
Figure 2. Development of 2PN and 3PN human embryos. (A) 2PN fertilised oocyte. (B) Same 2PN zygote during the first cleavage. (C) Diploid embryo on day 3 after fertilisation. (D) 3PN fertilised oocyte. (E) Same 3PN zygote during first cleavage with a tripolar spindle. (F) Triploid embryo on day 3 after fertilisation. Note similar morphological appearance on day 3 compared to 2PN in C (17).

POSTULATED MECHANISMS EXPLAINING SESQUIZYGOTIC TWINNING

DIANDRIC TRIPLOIDY

Dispermic fertilisation

Diandry is assumed to be the most likely cause of triploid embryos and manifests in approximately 5% of inseminated oocytes (6, 21). Diandric triploidy of dispermic origin refers to the fertilisation of one oocyte by two individual spermatozoa, thus resulting in two paternal pronuclei (Figure 3) (4). This form of triploidy is prevalent in normozoospermic males and may be confirmed by the presence of two Y chromosomes using cytogenetic analyses (22-24). Dispermic fertilisation occurs at a rate of 61.5% and has been demonstrated to account for 86% of triploid embryos following IVF (21).

To prevent polyspermy, the entrance of multiple sperm into the ooplasm, protective mechanisms such as the plasma membrane block and the oocyte cortical granule reaction exist (Figure 1). The plasma membrane block consists of a rapid electrical depolarization of the oolemma, right after its fusion with the sperm membrane (25). While the plasma membrane block has been extensively studied in sea urchins, the cellular signalling events involved in establishing membrane potential in humans remain poorly understood (25-27). The cortical reaction has been largely characterised across animal species and is known to occur due to the oscillation of calcium ions inside the oocyte (26). The subsequent exocytosis of cortical granules triggers a signalling cascade results in the hardening of the zona pellucida, among other features. This event prevents sperm binding after fertilisation, thus providing a definitive block to polyspermy (26, 28).

However, in the case of diandric triploidy these mechanisms fail, with the cause being attributable to multiple factors (4, 21, 29-31). Whereas explanations exist that establish direct connections between increased sperm concentrations and the incidence of polyspermy, alternative studies have shown an approximate 30% increase in polyspermy rate that seems to be associated with the dysregulation between the timing of ovulation and oocyte maturation (4, 24, 25, 31-33). In any case, the link between the incidence of 3PN and poor oocyte quality remains unclear. Consequently, whether the embryo is capable of correcting such errors during early development will require further study (34).

During development, the triploid state of the embryo may be maintained through the segregation of chromosomes resulting in a mixoploid embryo, consisting of diploid and triploid blastomeres (2n/3n) (Figure 2) (14). Alternatively, one haploid chromosome set may be eliminated; in this case, either a normal diploid zygote or a tripolar spindle may form. As a consequence of tripolar spindle formation, gross aneuploidy results, and embryonic development is unable to continue in most cases (35). However, the occasional survival of trisomics and uniparental disomies depend on mixoploidy and the proportion of diploid and triploid cells present in the embryo (35).

In the case of sesquizygosity, chimeric triploid-derived 2n embryonic cell populations may develop into mosaic/chimaeras or twins (35). This division of the zygote following dispermic fertilisation is described by a cytogenetic phenomenon defined as postzygotic diploidization of triploids (PDT), where individuals present with common maternal and distinct paternal genomes, yet display more similarity for some traits than seen in dizygotic twins (35). Thus, the most likely karyotype of sesquizygotic individuals would be 69, XXX, XXY, because cells of uniparental lineage are usually outcompeted by cells of biparental lineage; however, in very rare cases triploid embryos may convey XYY (Figure 3) (1, 23). Preimplantation Genetic Testing (PGT) may provide further insight into the intermediate between monozygotic and dizygotic twinning events (35), assessing for genotypic differences among circulating foetal DNA; whereas the absence of such differences is indicative of monozygotic twins, their presence suggests dizygosity (21). Since mosaic/chimeric and triploid embryos add to the complexity of PGT analysis, sesquizygosity further complicates this issue. Consequently, each sesquizygotic twinning event should be considered on a case-by-case basis.  

Dispermic & displospermic fertilisation schema

Figure 3. Proposed mechanisms of diandric triploidy. As described by Destouni et al., (36). (A) Following dispermic fertilisation, the parental genomes undergo separate segregation defined as heterogoneic cytokinesis. Subsequent tripolar spindle formation results in a triploid embryo and the development of cells with biparental lineage are preferred over androgenic (YY) uniparental lines to form a chimeric blastocyst. (B) Diplospermic fertilisation by a 2n spermatozoon usually incapable of producing a viable zygote (1, 36).

Diplospermic fertilisation

Diplospermic fertilisation occurs due to the fertilisation of one oocyte by a binucleate or diploid sperm (Figure 3) (37). This form of diandric triploidy is attributable to nondisjunction during either meiotic division of spermatogenesis, resulting in a sperm containing a diploid set of chromosomes (37, 38). While triploidy of diplospermic origin accounts for a portion of triploid embryos (8.3%), genetic analyses of 3PN zygotes following ICSI established that the presence of binucleate sperm occurs at a higher rate in oligosazoospermic, cryptoazoospermic and azoospermic males (33.3%), compared to approximately 0.3% in normozoospermic males (4, 37, 39). Initially, these prevalence rates may not seem to be of major concern, considering that binucleate sperm are usually incapable of fertilisation and of producing a viable zygote (4). Nevertheless, binucleate sperm may occasionally fertilise oocytes before completion of the first meiotic division or in the context of ICSI, where sperm are able to bypass the oocyte’s natural sperm selection mechanisms and form a triploid embryo (40-42).

Parthenogenetic oocyte activation

Diandry may also be the result of parthenogenetic oocyte activation, where embryo development commences without paternal genetic contribution, thus forming two diploid blastomeres (Figure 4) (43). Parthenogenetic activation is thought to result from a failed cytokinesis event, where in the absence of complete mitosis the genome of the cell is duplicated (4, 44, 45). In this circumstance, the fertilisation of a diploid two-blastomere embryo by two individual spermatozoa results in a triploid embryo (46). While this postulated mechanism provides a likely explanation for triploidy, studies in mouse models suggest that parthenogenetically activated oocytes are not capable of efficient sperm chromatin remodelling, and so they are unable to support zygote development (47).

Parthenogenetic activation of the oocyte resulting in diandric triploidy. Fertilisation of parthenogenetically activated oocyte by two individual haploid spermatozoa, resulting in a triploid embryo and chimeric blastocyst.

Figure 4. Parthenogenetic activation of the oocyte resulting in diandric triploidy. Fertilisation of parthenogenetically activated oocyte by two individual haploid spermatozoa, resulting in a triploid embryo and chimeric blastocyst.

DIGYNIC TRIPLOIDY

Triploidy of digynic origin refers to an extra maternal haploid set resulting from the fertilisation of a haploid oocyte by a spermatozoon. Digynic triploidy is predominant among early miscarriages and has been attributed to 50-60% of all spontaneous abortions (8). Triploidy in this instance is explained by failures during either meiotic division or as a consequence of polar body retention (Figure 5). Equal ratios of XXX and XXY with the absence of XYY cells imply embryos are of digynic origin (21).

Endoreduplication of the female nucleus

Endoreduplication of the female nucleus results in a ‘giant cell’ with a single haploid nucleus, and has been shown to accompany abnormal pronuclei formation (Figure 5) (45, 48). In this case, errors during the first or second meiotic division may explain digynic triploidy (49). Although triploidy of digynic origin due to endoreduplication is not entirely understood, the activation of complex downstream enzyme pathways as a requirement for the progression of meiotic development has been well-established (50). Consequently, the dysregulation of genetic or molecular factors involved in any stage of this process may result in post-meiotic cell errors, causing second polar body retention and subsequent triploidy (51).

Second polar body retention

Alternate explanations for digynic triploidy may be associated with second polar body retention (Figure 5). Studies using pericentromeric markers in triploid cell lines have demonstrated that this anomaly contributes to the majority of digynic triploid embryo cases (3%), and may be associated with failure during the second meiotic division, or due to disruption of the meiotic spindle following ICSI (10, 15). Several reports have associated second meiotic arrest with age-related infertility or errors during spindle formation (52, 53). However, due to the ethical issues surrounding the study of human fertilisation, the causative mechanisms of second polar body retention can only be observed during intervals of the ART procedures. As a result, these issues remain not well understood.

Proposed mechanisms of digynic triploidy. As described by Rosenbusch (3). (A) Digyny due to endoreduplication within the female pronucleus. Presence of an additional haploid maternal nucleus due to errors in the first meiotic division (not pictured) or the second meiotic division involving genome duplication without cell division. (B) Digyny due to non-extrusion of the second polar body (4).

Figure 5. Proposed mechanisms of digynic triploidy. As described by Rosenbusch (3). (A) Digyny due to endoreduplication within the female pronucleus. Presence of an additional haploid maternal nucleus due to errors in the first meiotic division (not pictured) or the second meiotic division involving genome duplication without cell division. (B) Digyny due to non-extrusion of the second polar body (4).

CONCLUSIONS

Twinning events provide insight into the cellular mechanisms occurring during embryogenesis that may influence triploidy. While in the context of ART well-established clinical and laboratory aspects may prevent the incidence of 3PN, in vitro rates may differ from in vivo rates due to various conditions for fertilisation, including treatment of infertile couples who may be predisposed to abnormal fertilisation events (17).

Whereas triploid embryos are usually an embryonic lethal event, extraordinary cases of dispermic fertilisation may provide explanations for the phenomenon of sesquizygotic twinning. As sesquizygosity is a rare event, ascertainment bias and difficulty defining criteria for such cases exist. However, a greater understanding of the aetiology and mechanisms involved in twinning events may aid management and possible prevention of such cases in the future.

REFERENCES

  1. Gabbett MT, Laporte J, Sekar R, Nandini A, McGrath P, Sapkota Y, et al. Molecular Support for Heterogonesis Resulting in Sesquizygotic Twinning. New England Journal of Medicine. 2019;380(9):842-9. (Cover Image)
  2. Westphal LM, Rosencrantz M, Behr B, Milki AA. Significance of one pronucleus before fertilization. Fertility and sterility. 2003;79(4):1031-3.
  3. Figueira RCS, Setti AS, Braga DPAF, Iaconelli A, Jr., Borges E, Jr. Prognostic value of triploid zygotes on intracytoplasmic sperm injection outcomes. Journal of assisted reproduction and genetics. 2011;28(10):879-83.
  4. Rosenbusch BE. Mechanisms giving rise to triploid zygotes during assisted reproduction. Fertility and sterility. 2008;90(1):49-55.
  5. Reichman DE, Jackson KV, Racowsky C. Incidence and development of zygotes exhibiting abnormal pronuclear disposition after identification of two pronuclei at the fertilization check. Fertility and sterility. 2010;94(3):965-70.
  6.  Fiorentino A, Tomasi G, Papale L, Montag M. The zygote. Human Reproduction. 2012;27(suppl_1):i22-i49.
  7. Li M, Zhao W, Xue X, Zhang S, Shi W, Shi J. Three pro-nuclei (3PN) incidence factors and clinical outcomes: a retrospective study from the fresh embryo transfer of in vitro fertilization with donor sperm (IVF-D). International journal of clinical and experimental medicine. 2015;8(8):13997-4003.
  8. McFadden DE, Robinson WP. Phenotype of triploid embryos. Journal of medical genetics. 2006;43(7):609-12.
  9. Kolarski M, Ahmetovic B, Beres M, Topic R, Nikic V, Kavecan I, et al. Genetic Counseling and Prenatal Diagnosis of Triploidy During the Second Trimester of Pregnancy. Medical archives (Sarajevo, Bosnia and Herzegovina). 2017;71(2):144-7.
  10. Van De Laar I, Rabelink G, Hochstenbach R, Tuerlings J, Hoogeboom J, Giltay J. Diploid/triploid mosaicism in dysmorphic patients. Clinical Genetics. 2002;62(5):376-82.
  11. Souter VL, Parisi MA, Nyholt DR, Kapur RP, Henders AK, Opheim KE, et al. A case of true hermaphroditism reveals an unusual mechanism of twinning. Human genetics. 2007;121(2):179-85.
  12. Carlson B. Human Embryology and Developmental Biology/Carlson BM–. New york: Elsevier Science (Mosby); 2004.
  13.  Yilmaz A, Zhang L, Zhang XY, Son WY, Holzer H, Ao A. Chromosomal complement and clinical relevance of multinucleated embryos in PGD and PGS cycles. Reproductive biomedicine online. 2014;28(3):380-7.
  14. Carson JC. A Comprehensive Examination of Human Triploidy and Diploid/Triploid Mixoploidy: University of Pittsburgh; 2009.
  15. Porter R, Han T, Tucker MJ, Graham J, Liebermann J, Sills ES. Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes. Journal of assisted reproduction and genetics. 2003;20(9):371-6.
  16. ASRM, ESHRE, editors. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human reproduction (Oxford, England); 2011 Jun: Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology.
  17. Kang HJ, Rosenwaks Z. Triploidy–the breakdown of monogamy between sperm and egg. The International journal of developmental biology. 2008;52(5-6):449-54.
  18. Rosen MP, Shen S, Dobson AT, Fujimoto VY, McCulloch CE, Cedars MI. Triploidy formation after intracytoplasmic sperm injection may be a surrogate marker for implantation. Fertility and sterility. 2006;85(2):384-90.
  19. Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature. 2014;508(7497):483-7.
  20. Jin F, Shi Y, Zhou F. [Influences of sperm quality and quantity on fertilization, cleavage rates and quality of embryos in in-vitro fertilization]. Zhonghua fu chan ke za zhi. 1998;33(1):28-30.
  21. Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Human reproduction (Oxford, England). 1997;12(2):321-7.
  22. Egozcue J, Blanco J, Vidal F. Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH). Hum Reprod Update. 1997;3(5):441-52.
  23. Pieters MHEC, Dumoulin JCM, Ignoul-Vanvuchelen RCM, Bras M, Evers JLH, Geraedts JPM. Triploidy after in vitro fertilization: Cytogenetic analysis of human zygotes and embryos. Journal of assisted reproduction and genetics. 1992;9(1):68-76.
  24. Rosenbusch B, Schneider M, Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Human reproduction (Oxford, England). 1997;12(10):2257-62.
  25. Gardner AJ, Evans JP. Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reproduction, Fertility and Development. 2005;18(2):53-61.
  26. Liu M. The biology and dynamics of mammalian cortical granules. Reproductive biology and endocrinology : RB&E. 2011;9:149-.
  27. Tosti E, Boni R. Electrical events during gamete maturation and fertilization in animals and humans. Human Reproduction Update. 2004;10(1):53-65.
  28. Gupta SK. Unraveling the intricacies of mammalian fertilization. Asian journal of andrology. 2014;16(6):801-2.
  29. Gilbert SF. Gamete Fusion and the Prevention of Polyspermy. NCBI: Sinauer Associates; 2000. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10033/.
  30. Sengoku K, Tamate K, Horikawa M, Takaoka Y, Ishikawa M, Dukelow WR. Plasma membrane block to polyspermy in human oocytes and preimplantation embryos. Journal of reproduction and fertility. 1995;105(1):85-90.
  31. Wang WH, Day BN, Wu GM. How does polyspermy happen in mammalian oocytes? Microscopy research and technique. 2003;61(4):335-41.
  32.    Burkart AD, Xiong B, Baibakov B, Jimenez-Movilla M, Dean J. Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. The Journal of cell biology. 2012;197(1):37-44.
  33.    Wolf DP, Byrd W, Dandekar P, Quigley MM. Sperm concentration and the fertilization of human eggs in vitro. Biology of reproduction. 1984;31(4):837-48.
  34.  Xia P. Biology of Polyspermy in IVF and its Clinical Indication. Current Obstetrics and Gynecology Reports. 2013;2(4):226-31.
  35. Golubovsky MD. Postzygotic diploidization of triploids as a source of unusual cases of mosaicism, chimerism and twinning. Human Reproduction. 2003;18(2):236-42.
  36.  Destouni A, Zamani Esteki M, Catteeuw M, Tsuiko O, Dimitriadou E, Smits K, et al. Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy. Genome research. 2016;26(5):567-78.
  37. Egozcue S, Blanco J, Vidal F, Egozcue J. Diploid sperm and the origin of triploidy. Human reproduction (Oxford, England). 2002;17(1):5-7.
  38. Fleischer J, Shenoy A, Goetzinger K, Cottrell CE, Baldridge D, White FV, et al. Digynic triploidy: utility and challenges of noninvasive prenatal testing. Clinical case reports. 2015;3(6):406-10.
  39. Macas E, Imthurn B, Keller PJ. Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI. Human reproduction (Oxford, England). 2001;16(1):115-20.
  40. Celik-Ozenci C, Jakab A, Kovacs T, Catalanotti J, Demir R, Bray-Ward P, et al. Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations. Human reproduction (Oxford, England). 2004;19(9):2052-9.
  41. Debois J-M. Selected Topics in Clinical Oncology: An In-depth Study of 18 Cancers Usually Neglected in Classical Textbooks: Springer Science & Business Media; 2012.
  42. Sánchez-Calabuig MJ, López-Cardona AP, Fernández-González R, Ramos-Ibeas P, Fonseca Balvís N, Laguna-Barraza R, et al. Potential Health Risks Associated to ICSI: Insights from Animal Models and Strategies for a Safe Procedure. Frontiers in public health. 2014;2:241-.
  43. Ferguson-Smith AC. Uniparental Inheritance. In: Brenner S, Miller JH, editors. Encyclopedia of Genetics. New York: Academic Press; 2001. p. 2096-9.
  44. Leng L, Ouyang Q, Kong X, Gong F, Lu C, Zhao L, et al. Self-diploidization of human haploid parthenogenetic embryos through the Rho pathway regulates endomitosis and failed cytokinesis. Scientific reports. 2017;7(1):4242-.
  45. Zielke N, Edgar BA, DePamphilis ML. Endoreplication. Cold Spring Harbor perspectives in biology. 2013;5(1):a012948-a.
  46. Golubovsky M. Paternal familial twinning: hypothesis and genetic/medical implications. Twin research : the official journal of the International Society for Twin Studies. 2002;5(2):75-86.
  47. Yang H, Shi L, Chen CD, Li J. Mice generated after round spermatid injection into haploid two-cell blastomeres. Cell research. 2011;21(5):854-8.
  48. Gläser B, Rosenbusch B, Brucker C, Schneider M. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy*. Human Reproduction. 2002;17(9):2388-93.
  49. Kaiser-Rogers KA, McFadden DE, Livasy CA, Dansereau J, Jiang R, Knops JF, et al. Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia. Journal of medical genetics. 2006;43(2):187-92.
  50. Blouin JL, Billieux MH, Petignat P, Vassilakos P, Dahoun S. Is genetic analysis useful in the routine management of hydatidiform mole? Human Reproduction. 2003;18(2):243-9.
  51. Wang Q, Racowsky C, Deng M. Mechanism of the chromosome-induced polar body extrusion in mouse eggs. Cell division. 2011;6:17-.
  52. Beall S, Brenner C, Segars J. Oocyte maturation failure: a syndrome of bad eggs. Fertility and sterility. 2010;94(7):2507-13.
  53. Wu XQ, Zhang X, Li XH, Cheng HH, Kuai YR, Wang S, et al. Translocation of classical PKC and cortical granule exocytosis of human oocyte in germinal vesicle and metaphase II stage. Acta pharmacologica Sinica. 2006;27(10):1353-8.