Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients.

BACKGROUND: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. RESULTS: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. CONCLUSIONS: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients.

Prospective assessment of follicular growth and the oocyte cohort after ovarian stimulation for fertility preservation in 90 cancer patients versus 180 matched controls.

A lower number of metaphase II oocytes eligible for vitrification after controlled stimulation in cancer patients has recently been reported, suggesting that cancer may impair the dynamics and quality of follicular growth. In this prospective, non-interventional study, the pattern of follicular growth and oocyte cohort after ovarian stimulation in cancer patients was analysed. Ninety cancer patients, recruited before starting chemotherapy, were compared with 180 time- and age-matched healthy controls undergoing intracytoplasmic sperm injection. Primary outcome was total number of metaphase II oocytes and metaphase II /total oocytes rate. Basal anti-Müllerian hormone levels (P < 0.05) and antral follicle count (P < 0.0001) were significantly lower in cancer patients. Recombinant FSH total dose was significantly higher in the cancer group (P < 0.0001). No differences were found in duration of stimulation, mean number of mature follicles on day of ovulation induction and total oocyte number after retrieval; the number of metaphase II oocytes retrieved (6.2 ± 4.7 versus 8.8 ± 4.2; P < 0.0001) and number of metaphase II oocytes-total oocytes ratio were significantly lower in cancer patients (56% versus 78%, P < 0.0001). Fewer metaphase II oocytes were eligible for vitrification and lower maturation rate in the cancer group.

First intention IVF protocol for polycystic ovaries: does oral contraceptive pill pretreatment influence COH outcome?

BACKGROUND: Morphological aspect of polycystic ovaries (PCO) is a very common finding in an IVF center population: this includes PCOS patients identified in 18-25% of the couples presenting with infertility and so called "sonographic PCO only" the prevalence of which has been estimated as high as 33% in asymptomatic patients. Finding the optimal first intention IVF protocol for polycystic ovaries patients is still challenging in order to improve the controlled ovarian hyperstimulation (COH) outcome while avoiding ovarian hyperstimulation syndrome (OHSS). It has been suggested that women with PCO would benefit from a longer period of pituitary down-regulation. The purpose of this study was to compare an extended duration of OCP pretreatment with a classic GnRH agonist protocol. METHODS: A single center prospective non-randomized study was performed from January 2009 to December 2010 in the Lille University Hospital including 113 women diagnosed with PCO(S) according to the Rotterdam ultrasonographic criteria and undergoing their first IVF attempt. Comprehensive hormonal and ultra-sonographic assessments were collected during COH in these patients. LH and androgen suppression and dynamics of follicular growth were compared between the two protocols as well as the COH outcome in terms of oocyte/embryo number and quality, implantation and pregnancy rates. RESULTS: No significant difference was observed between the two groups concerning dynamics of follicular growth and hormonal values. Clinical and ongoing pregnancy rates were significantly lower in the OCP group despite same oocyte and embryo quality. Nevertheless, the cumulative pregnancy rate did not differ between the two groups. The incidence of OHSS was not statistically significant. CONCLUSIONS: Extended duration of OCP pretreatment, as a first intention IVF protocol for PCO patients, does not improve the pattern of follicular growth nor the oocyte and embryo quality.

Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility.

BACKGROUND: Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared. METHODS: We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually. RESULTS: As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF. CONCLUSION: The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.