Identification of Premeiotic, Meiotic, and Postmeiotic Cells in Testicular Biopsies Without Sperm from Sertoli Cell-Only Syndrome Patients.

Sertoli cell-only syndrome (SCOS) affects about 26.3⁻57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3⁻7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions.

Prospective evaluation of early follicular ovarian stromal blood flow in infertile women undergoing IVF-ET treatment.

BACKGROUND: To evaluate the role of early follicular stromal flow studies in predicting ovarian response during IVF-ET treatment and to assess their correlation with ovarian reserve parameters and clinical pregnancy achievement. MATERIALS & METHODS: One hundred and sixty-eight consecutive and unselected infertile women undergoing their first IVF-ET treatment were included in the study. Basal ovarian reserve and stromal Doppler flow studies were performed in a natural cycle before starting treatment. Four Doppler indices were measured; peak systolic velocity (PSV), pulsatility index (PI), resistance index (RI) and systole/diastole ratio (S/D). Following completion of IVF-ET treatment Pearson's correlation analysis was performed to examine the correlation between Doppler indices, ovarian response, basal ovarian reserve parameters and clinical pregnancy achievement. RESULTS: A positive correlation was found between the number of ≥14 mm follicles on hCG day and PSV. The number of ≥14 mm follicles and retrieved oocytes had a significant negative correlation with RI and S/D ratio. As well, the number of fertilized oocytes had a significant negative correlation with S/D ratio. Absence of a Doppler signal in one or both ovaries was significantly higher in the women with poor response (31%) as compared to women with normal response (16%). In addition, RI correlated positively with basal FSH as well as FSH/LH ratio and negatively with AFC. The S/D ratio had a negative correlation with AFC (p = 0.027). A significant positive correlation between PSV, total ovarian volume (p = 0.011) and mean ovarian volume (p = 0.019) was detected. However, no correlation between all four Doppler indices and age was detected. Moreover, Doppler indices did not differ significantly between conception and non-conception cycles following IVF-ET treatment. CONCLUSIONS: Early follicular stromal Doppler signals is correlated with ovarian response as well as basal ovarian reserve parameters, but have no correlation with age neither with clinical pregnancy achievement in infertile women undergoing IVF-ET treatment.

A simple multivariate score could predict ovarian reserve, as well as pregnancy rate, in infertile women.

OBJECTIVE: To find a simple multivariate score that has the potential to predict ovarian reserve, as well as pregnancy rate, in infertile women. DESIGN: A prospective study. SETTING: A university-affiliated reproductive medicine unit. PATIENT(S): One hundred sixty-eight consecutive women undergoing their first IVF-ET treatment at our unit. INTERVENTION(S): Basal ovarian reserve studies, endocrine and sonographic, were performed before starting therapy. After completion of treatment, a logistic regression analysis was performed to examine which parameters significantly determined low ovarian reserve. These parameters were incorporated thereafter in a multivariate score to predict ovarian reserve, as well as clinical pregnancy rate. MAIN OUTCOME MEASURE(S): Low ovarian reserve defined as 14 was shown to be more accurate in predicting low ovarian reserve than age, day 3 FSH, or antral follicle count separately. Moreover, a score of >14 was shown to have a sensitivity of 88% and a specificity of 69% in predicting low ovarian reserve. More important, women with a score of >14 had significantly lower clinical implantation and pregnancy rates relative to women with a score of

Early and short follicular gonadotropin-releasing hormone antagonist supplementation improves the meiotic status and competence of retrieved oocytes in in vitro fertilization-embryo transfer cycles.

OBJECTIVE: To investigate whether early and short follicular administration of GnRH antagonist using the flexible protocol has the potential to improve IVF-ET clinical results. DESIGN: Prospective, controlled, randomized study. SETTING: University-affiliated assisted reproductive technology unit. PATIENT(S): Fifty-three consecutive infertile women were enrolled to the study and control groups. INTERVENTION(S): Both groups were treated with recombinant FSH and the flexible GnRH antagonist protocol. Women in the study group were additionally supplemented with three injections of GnRH antagonist (0.25 mg/d on days 1, 2, and 3 of the menstrual cycle). MAIN OUTCOME MEASURE(S): Hormonal milieu, oocyte meiotic status, competence for normal fertilization, cleavage, and clinical pregnancy rate. RESULT(S): Both groups had comparable baseline characteristics. The duration of recombinant FSH treatment was significantly longer in the study group as compared with the control group (10.9+/-3.1 and 9.7+/-1.3 days, respectively). The number of follicles>or=14 mm and E2 level on the day of hCG administration, number of retrieved oocytes, and endometrial thickness were similar between the two groups. However, the fertilization rate was significantly higher in the study as compared with the control group (85%+/-16% and 69%+/-24%, respectively). Moreover, the cumulative rate of mature first polar body oocytes was significantly higher in the study group as compared with the control group (93% and 85%, respectively). Concomitantly, day-3 FSH and LH levels after initiation of treatment were significantly lower in the study as compared with the control group (6.1+/-2.4 mIU/mL vs. 7.2+/-1.9 mIU/mL and 2.4+/-1.6 mIU/mL vs. 5.6+/-2.7 mIU/mL, respectively). CONCLUSION(S): Early and short follicular GnRH antagonist supplementation using flexible GnRH antagonist treatment improves the meiotic status and competence of retrieved oocytes. It seems that early and short pituitary shutdown has the potential to improve clinical results in IVF-ET GnRH antagonist cycles.

First polar body and nucleolar precursor body morphology is related to the ovarian reserve of infertile women.

This study was undertaken in order to gain insight into the morphology of the first polar body (PB1) and the two pronuclei (2PN) in ICSI patients, specifically the nucleolar precursor bodies (NPB). Whether early abnormalities in these structures are related to the ovarian reserve of infertile women was also studied. Eighty consecutive infertile women were prospectively investigated throughout their first ICSI cycles. Basal ovarian reserve studies were performed in all women. Cycles were evaluated with respect to PB1 and 2PN morphology of the transferred embryos. Cycles that had at least one transferred embryo with normal PB1 and 2PN morphology had significantly better basal ovarian reserve parameters compared with cycles in which all transferred embryos had abnormal PB1 and 2PN morphology. Moreover, the normal morphology group performed significantly better throughout the ovarian stimulation, compared with the abnormal morphology group. Furthermore, the clinical implantation and pregnancy rates were significantly higher in the normal compared with the abnormal morphology group, corresponding to 20.7% versus 10.6% and 42.4% versus 18.2%, respectively. The study concluded that the morphology of the PB1 in metaphase II oocytes as well as that of the NPB within the 2PN zygotes seems to be related to the ovarian reserve in infertile women.

The effects of prostaglandin F2alpha treatment on peripheral-type benzodiazepine receptors in the ovary and uterus during pseudopregnancy of rats.

A previous study by us indicated that peripheral-type benzodiazepine receptor (PBR) density may be increased in the ovaries and uterus of pregnant rats (Weizman R, Dagan E, Snyder SH, Gavish M. Impact of pregnancy and lactation on GABAA receptor and central-type and peripheral-type benzodiazepine receptors. Brain Res 1997;752:7-14). In the present study, the effects of prostaglandin F2alpha (PGF2alpha) on PBR density in the ovary and uterus of pseudopregnant rats were assayed. Pseudopregnancy was induced on day 29 post-partum (PP) by s.c. injection of 50IU pregnant mare serum gonadotropin (PMSG) and 3 days later by s.c. injection of 20IU human chorionic gonadotropin (hCG). PBR ligand binding density was assayed with the specific PBR ligand [3H]PK 11195. A two-fold increase in ovarian PBR density was observed 2 days after hCG administration compared with vehicle control rats and this effect was maintained for 3 weeks. In the uterus, a three-fold increase in PBR density was observed and this increase was maintained for 1 week after hCG administration. Pseudopregnancy did not appear to affect renal PBR density or affinity. Treatment with PGF2alpha, which causes luteolysis, resulted in an approximately 50% reduction of PBR density in the ovaries of pseudopregnant rats at day 53 PP compared to pseudopregnant control rats. Treatment with indomethacin, which prevents the formation of PGF2alpha, caused the PBR density in the uterus of pseudopregnant rats at day 53 PP to be twice as high as in pseudopregnant control rats. All the above treatments did not affect the affinity of [3H]PK 11195 to ovarian and uterine PBR. These data suggest that PBR density in corpora lutea and uterus during pseudopregnancy is regulated by PGF2alpha.

Poor oocyte retrieval is a manifestation of low ovarian reserve.

Women with complete absence of oocytes during retrieval, as well as those with less than the 10th percentile of the expected number of oocytes retrieved, have clear manifestations of low ovarian reserve. It seems that this occurrence is a gradual biological phenomenon related to the basic pathophysiology of ovarian aging.

Preimplantation genetic diagnosis for a couple with recurrent pregnancy loss and triploidy.

BACKGROUND: Triploidy may arise from fertilization of a mature haploid egg by two haploid sperm or by failure of meiotic divisions yielding a diploid gamete. We encountered a couple with habitual abortion, in which the last two fetuses were documented as viable triploid. METHODS: To avoid dispermic penetration and development of abnormal preembryos, insemination was done by intracytoplasmic sperm injection (ICSI) followed by fluorescence in situ hybridization (FISH) of biopsied blastomeres. RESULTS: Tests of the husband's spermatozoa by FISH, revealed that only 2-3% of the sperm were disomic for chromosomes 16, 13, 21, X, and Y. No triple disomy was detected among chromosomes 16, 13 and 21, which makes it very unlikely that triploidy resulted from diploid spermatozoa. Following a controlled ovulation induction protocol, low quality oocytes with immature cumuli were revealed. After ICSI, five eggs became two pronuclei (2PN) zygotes and none of the other eggs developed a 3PN zygote. FISH was performed on chromosomes 16 and 21 in four preembryos developed to a 6-8 cell stage. Aneuploidy or mosaicism for each of these chromosomes was detected in one preembryo and later in two disaggregated blastocysts. FISH failed in one preembryo that became atretic after biopsy. CONCLUSIONS: Although this case was unsuccessful in achieving embryo transfer and normal pregnancy, we detected many abnormal morphological features in the oocytes and chromosomal abnormalities in the cleaving preembryos. This protocol can be proposed to patients with recurrent pregnancy loss associated with chromosomal abnormalities in the fetus.

Improved development of very-poor-quality human preembryos by coculture with human fallopian ampullary cells.

PURPOSE: To determine whether a confluent culture of fallopian ampullary epithelial cells, taken from women at the end of their reproductive life, is capable of rescuing very-poor-quality preembryos from cleavage arrest and/or degeneration. METHODS: Human preembryos. rejected for transfer or freezing because of very poor quality, and arrested within 24 h of cleavage, were cultured for 5 days in medium alone or over a confluent culture of fallopian ampullary epithelia] cells. Morphological criteria were utilized to assess preembryo degeneration and stage of development. RESULTS: The described coculture rescued preembryos from degeneration, enhancing development to the blastocyst stage 2.2-fold, compared with cultures in medium alone. Furthermore, fully expanded and hatching blastocysts were observed only under coculture conditions. CONCLUSIONS: Very-poor-quality human preembryos may be rescued from degeneration, and their growth and development dramatically improved, when cocultured with a confluent culture of fallopian ampullary epithelial cells.