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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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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Haplotype analysis of chemokine CXCL12 polymorphisms and susceptibility to premature ovarian failure in Chinese women

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Binbin Wang1,2,†, Peisu Suo1,2,†, Beili Chen3, Zhaolian Wei2, Lu Yang3, Sirui Zhou1,2, Jing Wang1,2, Yunxia Cao3 and Xu Ma1,2,4,*

1Center for Genetics, National Research Institute for Family Planning, 12, Dahuisi Road, Haidian, Beijing 100081, China

2Peking Union Medical College, Beijing, China

3Reproductive Medicine Center, First Affiliated Hospital, Anhui Medical University, Hefei, China

4World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing, China *Correspondence address. Tel: +86-10-62176870; Fax: +86-10-62179151; E-mail: genetic{at}263.net.cn?† These authors contributed equally to the work.

Received June 5, 2010. Revision received December 19, 2010. Accepted January 5, 2011. BACKGROUND Chemokine (C-X-C motif) ligand 12 (CXCL12/stromal cell-derived factor 1) has been suggested to play an essential role in primordial germ cell migration, colonization and survival, and in the primordial to primary follicle transition. This study was performed to investigate an association of polymorphisms in CXCL12 with the risk of premature ovarian failure (POF) in Chinese patients.

METHODS Tagging single nucleotide polymorphisms (SNPs) were selected using the Chinese HapMap database. Five SNPs (rs4948878, rs1801157, rs266087, rs266093 and rs1029153) were genotyped by direct sequencing in 111 patients with POF and 183 healthy controls recruited from the First Affiliated Hospital, Anhui Medical University, China.

RESULTS Compared with controls, there were significantly higher frequencies of the rs1801157 A allele and haplotype C-T-A-T-T in cases with POF [P = 6.38E-07, odds ratio (OR) = 3.10, 95% confidence interval (CI) 1.955–4.890 by allele; P = 7.0E-04, OR = 2.39, 95% CI 1.43–3.97 by haplotype]. No differences were observed for the other four SNPs between POF cases and controls.

CONCLUSIONS A strong association between a CXCL12 polymorphism and POF was established in Chinese patients, suggesting that CXCL12 might be a new candidate gene involved in POF. The A allele of CXCL12 polymorphism rs1801157 is a possible risk factor for developing POF. However, further independent studies are necessary to confirm our findings.

© 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): 950-954. doi: 10.1093/humrep/der001 First published online: February 4, 2011

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