Jean François Guerin

Normal pregnancies and live births after autograft of frozen-thawed hemi-ovaries into ewes

OBJECTIVE To evaluate long-term outcome of autotransplantation of cryopreserved hemi-ovaries into ewes. DESIGN Animal study. SETTING University fertility center, Hospices Civils de Lyon; and Ecole Nationale Vétérinaire de Lyon. PATIENT(S) Grivette ewes. INTERVENTION(S) Six hemi-ovaries from 6 ewes aged 6 to 12 months were frozen with a slow cooling protocol using 2 M of dimethyl sulfoxide as cryoprotectant. After dissection of the medulla, the hemi-ovarian cortex was stored at -196 degrees C in liquid nitrogen. Freezing procedure was performed with a programmable freezer. Semiautomatic seeding was performed before crystallization. Four to 6 weeks after the first laparotomy, the left ovary was removed and the frozen-thawed hemi-ovary was sutured. MAIN OUTCOME MEASURE(S) Mean plasma concentrations of FSH, LH, and progesterone after autotransplantation of frozen-thawed hemi-ovary. Ultrasonography was done to confirm pregnancy. Blood samples were collected weekly to measure FSH, LH, and progesterone. After the first birth, the autografted ovary was removed for histologic examination. RESULT(S) Plasma progesterone concentration increased in a regular manner in all ewes except one 4 weeks after the graft. Concentrations of FSH and LH did not reach the menopausal level. Four pregnancies occurred, from which 6 lambs were born. The first delivery of a normal lamb occurred after 135 days of gestation; the lamb died immediately after birth. The second delivery of two normal lambs occurred after 130 days of gestation. A caesarean section was performed on the third pregnant ewe the 110th days of gestation because the ewe had a vaginal prolapsus. The two normal lambs and the ewe died after surgery. The fourth birth of a normal lamb occurred after 132 days of gestation. Histologic examination of the grafted frozen-thawed ovary showed a regressing corpus luteum and few primordial and antral follicles. CONCLUSION(S) These four pregnancies in a ewe model may indicate that women who undergo preservation of their ovaries before chemotherapy or radiotherapy can have successful pregnancy.

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols

OBJECTIVE To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S) Lambs 5 to 6 months of age. INTERVENTION(S) Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. MAIN OUTCOME MEASURE(S) Follicular mortality and histologic structure. RESULT(S) For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S) Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep

OBJECTIVE To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. DESIGN Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. SETTING Fertility clinic in a large university hospital. ANIMALS Five- to 6-month-old lambs. INTERVENTION(S) Two-millimeter-thick slices of hemi-ovary cortex were prepared. MAIN OUTCOME MEASURE(S) Histological structure and DNA fragmentation. RESULT(S) In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. CONCLUSION(S) The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.

Quantitation by image analysis of global DNA methylation in human spermatozoa and its prognostic value in in vitro fertilization: a preliminary study

OBJECTIVE To determine the relationship between sperm DNA methylation level and sperm characteristics and pregnancy rates. DESIGN Prospective study. Quantitation by image analysis of DNA methylation in sperm nucleus. SETTING Department of Reproduction Biology, Edouard Herriot Hospital, Lyon, France. PATIENT(S) Infertile couples undergoing IVF-ET. INTERVENTION(S) The immunostaining of 5 methyl-cytosine was performed on the spare sperm suspension that was used for an assisted reproduction technology procedure. MAIN OUTCOME MEASURE(S) Sperm characteristics according to World Health Organization criteria, sperm motility parameters with computer-assisted semen analysis, sperm DNA methylation level, and heterogeneity index (HI). RESULT(S) Sperm DNA methylation level and HI are correlated with sperm DNA characteristics. HI is negatively correlated with fertilization rate; sperm DNA methylation level is correlated with pregnancy rate. CONCLUSION(S) The DNA methylation level in human spermatozoa could be a new approach to evaluating the ability of spermatozoa to fertilize and lead to normal embryo development.

The cryopreservation of ovarian tissue: uses and indications in veterinary medicine

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.