Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue

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Mona Sheikhi1,*, Kjell Hultenby2, Boel Niklasson1, Monalill Lundqvist1 and Outi Hovatta1

1Division of Obstetrics and Gynaecology, Department of Clinical, Science, Technology and Intervention, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

2Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, SE 141 86 Stockholm, Sweden *Correspondence address. Tel: +46-8-5858-0000; Fax: +46-8-58587250; E-mail: mona.sheikhi{at}karolinska.seReceived March 17, 2010. Revision received November 10, 2010. Accepted November 22, 2010. BACKGROUND Cancer therapy is one of many conditions which may diminish the ovarian reserve. Banking of human ovarian tissue has become an option for the preservation of female fertility. We have shown that vitrification is an excellent method to cryopreserve ovarian tissue. To carry out vitrification in a clinical setting, we have developed a clinical grade closed system to avoid direct contact of ovarian tissue with liquid nitrogen.

METHODS Ovarian tissue was obtained by biopsy from 12 consenting women undergoing Caesarean section. Tissues were vitrified in cryotubes, using dimethyl sulphoxide, 1,2-propanediol, ethylene glycol and polyvinylpyrrolidon as cryoprotectants. Non-vitrified and warmed-vitrified tissue was compared by light and electron microscopic morphology of the follicles within the tissues.

RESULTS We did not see any differences in the light or electron microscopic ultrastructure of oocytes between non-vitrified and vitrified tissues. No irreversible subcellular alterations in vitrified tissues were seen.

CONCLUSIONS The ultrastructure of follicles within the vitrified human ovarian tissue was well preserved using cryotube in a closed vitrification system to avoid direct contact of liquid nitrogen. The system is compatible with the European tissue directive.

© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

This ArticleHum. Reprod. (2011) 26 (3): 594-603. doi: 10.1093/humrep/deq357 First published online: January 8, 2011

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Efficiency of polarized microscopy as a predictive tool for human oocyte quality

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B. Heindryckx*, S. De Gheselle, S. Lierman, J. Gerris and P. De Sutter

Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium *Correspondence address. Tel: +32-9-332-4748; Fax: +32-9-332-4972; E-mail: bjorn.heindryckx{at}ugent.beReceived September 1, 2010. Revision received October 29, 2010. Accepted December 3, 2010. BACKGROUND Conflicting results have been reported regarding the use of polarized microscopy as a predictive tool for human oocyte quality.

METHODS Oocytes from 121 ICSI cycles were analysed with polarized microscopy. Both qualitative (spindle presence) and quantitative (retardance) data were correlated to the key assisted reproduction technology outcome parameters. Second, polarized microscopy was applied on in vitro matured (IVM) oocytes from germinal vesicle oocytes that matured after 24 or 48 h and from metaphase I oocytes matured after 3 or 24 h. These data were correlated with confocal analysis of spindle-chromosome complex.

RESULTS Spindles were detected in 82% of in vivo matured oocytes and in 64% adjacent to the first polar body (PB). Fertilization rate was higher in oocytes with a visible spindle (P = 0.0002). In patients aged over 35 years, the percentage of a visible spindle and mean spindle retardance was lower than in younger patients (P < 0.03). A higher number of spindles were located adjacent to the first PB in IVM matured oocytes (94%) versus in vivo matured oocytes (P < 0.0001). Confocal imaging revealed that spindle absent IVM metaphase II (MII) oocytes had a higher degree of aberrant spindle and chromosomal configurations versus IVM MII oocytes with a visible spindle (P = 0.002).

CONCLUSIONS Oocytes with absent spindles were associated with lower fertilization rates and advanced female age. Other important outcome parameters (embryo quality, pregnancy rates) were not correlated to spindle nor zona inner layer analysis. Interestingly, confocal imaging showed that polarized microscopy might be used as a qualitative predictive tool of human oocyte quality but no correlation could be demonstrated with quantitative polarized microscopy.

© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This ArticleHum. Reprod. (2011) 26 (3): 535-544. doi: 10.1093/humrep/deq376 First published online: January 12, 2011

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Cellular and genetic analysis of oocytes and embryos in a human case of spontaneous oocyte activation

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Catherine M.H. Combelles1,*, William G. Kearns2, Janis H. Fox3 and Catherine Racowsky3

1Biology Department, Middlebury College, Middlebury, VT 05753, USA

2Shady Grove Center for Preimplantation Genetics, Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

3Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA *Correspondence address. E-mail: ccombell{at}middlebury.eduReceived October 4, 2010. Revision received November 18, 2010. Accepted November 25, 2010. Unusual and consistent defects in infertility patients merit attention as these may indicate an underlying genetic abnormality, in turn necessitating tailored management strategies. We describe a case of repeated early pregnancy loss from in vivo conceptions, followed by cancelled embryo transfers after one IVF and one ICSI/PGD cycle. Following the unexpected presence of cleaved embryos at the fertilization check in the first IVF attempt, oocytes and embryos were subsequently analyzed in an ICSI/PGD case. Part of the oocyte cohort was fixed at retrieval for a cellular evaluation of microtubules, microfilaments and chromatin. The remaining oocytes were injected with sperm, and resultant embryos were biopsied for genetic analysis by fluorescence in situ hybridization (FISH), single-nucleotide polymorphism (SNP) microarray for 23 chromosome pairs, as well as with PCR for sex chromosomes. The presence of interphase microtubule networks and pronuclear structures indicated that oocytes were spontaneously activated by the time of retrieval. FISH revealed aneuploidy in all seven blastomeres analyzed, with all but two lacking Y chromosomes. Microarray SNP analysis showed an exclusively maternal origin of all blastomeres analyzed, which was further confirmed by PCR. From our multi-faceted analyses, we conclude that spontaneous activation, or parthenogenesis, was probably the pathology underlying our patient's recurrent inability to maintain a normal pregnancy. Such analyses may prove beneficial not only in diagnosing case-specific aberrations for other patients with similar or related failures, but also for furthering our general understanding of oocyte activation.

© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This ArticleHum. Reprod. (2011) 26 (3): 545-552. doi: 10.1093/humrep/deq363 First published online: January 11, 2011

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The association of maternal factors with delayed implantation and the initial rise of urinary human chorionic gonadotrophin

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A.M.Z. Jukic1,*, C.R. Weinberg2, D.D. Baird1 and A.J. Wilcox1

1Epidemiology Branch, National Institute of Environmental Health Sciences, PO Box 12233, MD A3-05, Durham, NC 27709, USA

2Biostatistics Branch, National Institute of Environmental Health Sciences, PO Box 12233, MD A3-05, Durham, NC 27709, USA *Correspondence address. E-mail: jukica{at}niehs.nih.govReceived August 4, 2010. Revision received January 5, 2011. Accepted January 11, 2011. BACKGROUND Late implantation and the pattern of early rise in hCG have been associated with early pregnancy loss. We explored factors that might be predictive of these markers of poor embryonic health in spontaneously conceived pregnancies.

METHODS Participants in the North Carolina Early Pregnancy Study collected daily first-morning urine specimens while attempting to conceive. Samples were assayed for estrogen and progesterone metabolites (to identify day of ovulation) and hCG (to detect conception). Data were available for 190 pregnancies, 48 of which ended in early loss (within 6 weeks of the last menstrual period). We used logistic regression to identify characteristics associated with late implantation (=10 days post-ovulation). For pregnancies surviving at least 6 weeks (n= 142), we used linear mixed models to identify factors associated with variations in hCG rise in the first 7 days from detection.

RESULTS Later implantation was associated with current maternal smoking [odds ratio (OR): 5.7; 95% confidence interval (CI): 1.1–30] and with oocytes that were likely to have been fertilized late in their post-ovulatory lifespan (OR: 5.1; CI: 1.9–16). Older women had a faster rise in hCG (P= 0.01), as did women who had relatively late menarche (P for trend = 0.02). Women exposed in utero to diethylstilbestrol showed an unusual pattern of slow initial hCG rise followed by a fast increase, a pattern significantly different from that of unexposed women (P= 0.002).

CONCLUSIONS Although limited by small numbers and infrequent exposures, our analyses suggest that a woman's exposures both early in life and at the time of pregnancy may influence early development of the conceptus.

Published by Oxford University Press 2011This ArticleHum. Reprod. (2011) 26 (4): 920-926. doi: 10.1093/humrep/der009 First published online: February 2, 2011

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Aberrant spindle structures responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially

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B. Heindryckx1,*, S. Lierman1, C.M. Combelles2, C.A. Cuvelier3, J. Gerris1 and P. De Sutter1

1Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium

2Biology Department, Middlebury College, Middlebury, VT, USA

3Department of Pathology, Ghent University Hospital, Ghent, Belgium *Correspondence address. Tel: +32-9-332-4748; Fax: +32-9-332-4972; E-mail: bjorn.heindryckx{at}ugent.beReceived September 7, 2010. Revision received November 22, 2010. Accepted December 20, 2010. BACKGROUND In some couples, not all retrieved oocytes mature, even after prolonged in vitro culture. The underlying mechanisms are not known, although ionophore treatment may alleviate metaphase I (MI) arrest in some mouse strains. We attempted to induce first polar body (PB) extrusion and fertilization using assisted oocyte activation (AOA) after ICSI in maturation-resistant human MI oocytes.

METHODS Four ICSI patients are described in this retrospective study. A pilot study tested the calcium ionophore ionomycin (10 µM) on donated MI oocytes from patients with a normal number of metaphase II (MII) oocytes. Subsequently, ionomycin was used to induce first PB extrusion in two patients showing maturation-resistant MI oocytes. AOA, by calcium injection and ionomycin exposure, was applied when mature oocytes were available. Oocytes were analysed by polarized microscopy and immunostaining.

RESULTS Ionomycin induced the first PB extrusion in MI oocytes from patients with a normal number of retrieved MII oocytes, while extended in vitro culture failed to achieve the MII stage. Similarly, ionomycin induced first PB extrusion in one of two patients with recurrent maturation-resistant MI oocytes. Use of ICSI combined with AOA on MII oocytes matured in vitro or in vivo resulted in failed or abnormal fertilization with no further embryo cleavage potential. Highly abnormal spindle and chromosome configurations were observed in MI maturation-resistant oocytes, in contrast to control MI oocytes.

CONCLUSIONS Ionophore induced first PB extrusion in MI oocytes from patients without maturation arrest but to a lower extent in maturation-resistant MI oocytes. Immunofluorescence staining and confocal analysis revealed, for the first time, highly abnormal spindle/chromosomal structures that may be responsible for this maturation arrest.

© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This ArticleHum. Reprod. (2011) 26 (4): 791-800. doi: 10.1093/humrep/deq400 First published online: January 17, 2011

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Decidual HtrA3 negatively regulates trophoblast invasion during human placentation

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Harmeet Singh1,*, Yaeta Endo2 and Guiying Nie1

1Prince Henry's Institute of Medical Research, PO Box 5152, 246 Clayton Road, Clayton, VIC 3168, Australia

2Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan *Correspondence address. Fax: +61-3-9594-6125; E-mail: harmeet.singh{at}princehenrys.orgReceived November 17, 2010. Revision received December 23, 2010. Accepted January 12, 2011. BACKGROUND Controlled trophoblast invasion into the maternal decidua (interstitial invasion) is important for placental development. Factors secreted by the maternal decidual cells and the extravillous trophoblast can influence trophoblast invasion and abnormalities in the invasion process may lead to pregnancy complications. Serine protease HtrA3 is highly expressed in the decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy, and in most trophoblast cell types, apart from the invading interstitial trophoblast during the first trimester. HtrA3 and its family members are down-regulated in a number of cancers and are proposed as tumour suppressors. The current study aimed to investigate whether HtrA3 is secreted by decidual cells, and whether inhibiting such secretion alters trophoblast invasion.

METHODS AND RESULTS Human endometrial stromal cells (HESCs) were decidualized with estradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate. Real-time RT–PCR, western blotting and immunocytochemistry confirmed that HtrA3 mRNA and protein expression increased during decidualization. HtrA3 was also detected in the conditioned media (CM) of the decidualized HESCs, confirming its secretion. For functional studies, a protease-inactive mutant form of HtrA3 which was previously confirmed to be a dominant-negative inhibitor was produced using wheat germ cell-free technology. CM from decidualized HESCs significantly suppressed invasion of trophoblast HTR-8 cells (P < 0.01), whereas inhibition of HtrA3 in this CM by exogenous HtrA3 mutant resulted in increased trophoblast HTR-8 cell invasion (P < 0.001).

CONCLUSIONS These results strongly support the hypothesis that decidual HtrA3 negatively regulates trophoblast invasion.

© The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This ArticleHum. Reprod. (2011) 26 (4): 748-757. doi: 10.1093/humrep/der019 First published online: February 14, 2011

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