Ettie Maman

Luteal phase oocyte retrieval and in vitro maturation is an optional procedure for urgent fertility preservation

OBJECTIVE To compare the results of in vitro maturation (IVM) of oocytes for fertility preservation performed during the luteal phase of the cycle with those of IVM performed during the follicular phase. DESIGN Retrospective chart review (August 2007 to June 2009). SETTING Academic tertiary referral fertility center. PATIENT(S) Cancer patients who underwent treatment for fertility preservation. INTERVENTION(S) IVM treatment during either luteal or follicular phase. MAIN OUTCOME MEASURE(S) Number of oocytes, maturation and fertilization rates, and number of oocytes and embryos that were frozen. RESULT(S) Eighteen cancer patients underwent IVM fertility preservation, five in their luteal phase and 13 in their follicular phase. The baseline characteristics of both groups were similar. There were no significant differences in the number of retrieved oocytes, maturation rates, fertilization rates, or the total number of oocytes and embryos that were cryopreserved. CONCLUSION(S) These results suggest that IVM during the luteal phase can be offered to patients as an optional treatment for urgent fertility preservation when there is insufficient time for conventional follicular phase oocyte retrieval before chemotherapy must be initiated.

Low expression of COX-2, reduced cumulus expansion, and impaired ovulation in SULT1E1-deficient mice

The SULTlEl‐encoded estrogen sulfo‐transferase (EST) catalyzes sulfation of estrogen, resulting in its inactivation. Reduced fertility observed in SULT1E1 knockout (KO) female mice has previously been attributed to the deleterious effect of chronic exposure to high levels of circulating estrogen on placental function. We herein suggest that, in addition to placental dysfunction, this phenotype demonstrates that an excess of estrogen impairs ovulation. The role of SULT1E1 in ovulation is suggested by the substantially low ovulatory response in hCG‐treated SULT1E1 KO mice;a similar effect was observed when 17β‐estradiol was administered to wild‐type (WT) females. The normal rate of ovulation in SULT1E1 KO females may be restored by PGE2. Along this line, ovaries of human Chorionic Gonadotropin (hCG)‐treated SULT1E1 KO mice expressed low levels of cyclooxy‐genase‐2 (COX‐2) and its downstream TSG6;moreover, their ovaries contained a reduced number of expanded cumuli. Our results demonstrate, for the first time, that estrogen inactivation may allow the expression of COX‐2 and subsequent cumulus expansion, enabling normal ovulation. Our findings may be applied to novel treatments of human ovulatory failure.—Gershon, E., Hourvitz, A., Reikhav, S., Maman, E., Dekel, N. Low expression of COX‐2, reduced cumulus expansion, and impaired ovulation in SULT1E1‐deficient mice. FASEB J. 21, 1893–1901 (2007)

Does local injury to the endometrium before IVF cycle really affect treatment outcome? Results of a randomized placebo controlled trial

Aim: To evaluate the effect of local injury to the endometrium during spontaneous menstrual cycles before in vitro fertilization (IVF) treatment on implantation and pregnancy rates in women with recurrent implantation failure (RIF). Methods: In a prospective randomized controlled trial (RCT), a total of 36 patients, with RIF undergoing IVF, were randomized to two groups. In 18 patients, endometrial biopsies were performed using a pipelle curette on days 9–12 and 21–24 of the menstrual cycle preceding IVF treatment. In 18 control patients, a cervical pipelle was performed. Results: The implantation rate (2.08% versus 11.11%; p = 0.1), clinical (0% versus 31.25%; p < 0.05) and live births rates (0% versus 25%; p = 0.1) were lower in the experimental group compared with controls. Conclusion: Our RCT did not find any benefit from local injury to the endometrium in women with a high number of RIFs. Further studies are warranted to better define the target population of patients who may benefit from this procedure.

High expression of luteinizing hormone receptors messenger RNA by human cumulus granulosa cells is in correlation with decreased fertilization.

OBJECTIVE To elucidate the LH receptor (LHR) expression patterns in human granulosa cells (GCs) from antral to preovulatory stages, and to investigate a correlation to oocyte function. DESIGN Luteinized preovulatory GCs were obtained from preovulatory follicles aspirated during IVF (≥ 17 mm). The GCs from small- (<10 mm) and medium-sized (10-15 mm) follicles were obtained during in vitro maturation (IVM) procedures. Cumulus GCs were obtained during oocyte denudation for intracytoplasmatic sperm injection (ICSI) procedures (IVF). SETTING Referral center. PATIENT(S) Seventy IVF patients and 20 IVM patients. INTERVENTION(S) GC collection. MAIN OUTCOME MEASURE(S) The LHR expression levels in mural and cumulus GCs of different follicular sizes and their correlation to oocyte outcome. RESULT(S) The LHR expression increased with follicle size and was higher in mural GCs compared with cumulus cells. The LHR expression in cumulus GCs from preovulatory follicles was higher in metaphase II (MII) oocytes than in metaphase I or germinal vesicle oocytes (IVF). Unexpectedly, higher expression of LHR in cumulus GCs of MII oocytes correlated with decreased fertilization rates. CONCLUSION(S) The LHR expression in small follicles obtained in IVM suggests a role for hCG administration during IVM procedures. Overexpression of LHR in cumulus GCs of MII oocytes may signal malfunction of oocytes and low fertilization capacity.

Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation

Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.

Localization of luteinizing hormone receptor protein in the human ovary

The luteinizing hormone receptor (LHR) plays a pivotal role during follicular development. Consequently, its expression pattern is of major importance for research and has clinical implications. Despite the accumulated information regarding LHR expression patterns, our understanding of its expression in the human ovary, specifically at the protein level, is incomplete. Therefore, our aim was to determine the LHR protein localization and expression pattern in the human ovary. We examined the presence of LHR by immunohistochemical staining of human ovaries and western blots of mural granulosa and cumulus cells aspirated during IVF treatments. We were not able to detect LHR protein staining in primordial or primary follicles. We observed equivocal positive staining in granulosa cells and theca cells of secondary follicles. The first appearance of a clear signal of LHR protein was observed in granulosa cells and theca cells of small antral follicles, and there was evidence of increasing LHR production as the follicles mature to the pre-ovulatory stage. After ovulation, LHR protein was ubiquitously produced in the corpus luteum. To confirm the expression pattern in granulosa cells and cumulus cells, we performed western blots and found that LHR expression was stronger in granulosa cells than in cumulus cells, with the later demonstrating low, but still significant, amounts of LHR protein. In summary, we conclude that LHR protein starts to appear on granulosa cells and theca cells of early antral follicles, and low but significant expression of LHR exists also in the cumulus cells. These results may have implications for the future design of clinical protocols and culture mediums for in vitro fertilization and especially in vitro maturation of oocytes.

Anti-Müllerian hormone is highly expressed and secreted from cumulus granulosa cells of stimulated preovulatory immature and atretic oocytes

This study investigated anti-Müllerian hormone (AMH) expression and secretion from cumulus granulosa cells (GC) and steroidogenesis in follicular fluids (FF) with relation to oocyte maturational stages and fertilization capacity in large preovulatory follicles. This prospective study included 53 ovulatory women undergoing intracytoplasmic sperm injection. FF and cumulus GC from 140 large preovulatory follicles were individually obtained during oocyte retrieval. Main outcome measures were oocyte maturation, fertilization and embryo quality. FF were assayed for AMH, progesterone, 17β-oestradiol and testosterone. Cumulus GC were assayed for AMH mRNA expression. AMH mRNA expression and secretion in cumulus GC in preovulatory follicles containing germinal-vesicle (GV) and metaphase-I (MI) oocytes were significantly higher than follicles containing MII oocytes (P<0.01 and P<0.0001, respectively). In addition, FF AMH concentrations from atretic oocytes were significantly higher than from MII oocytes. No correlation was found between AMH expression and secretion to fertilization or embryo quality. FF of MI and GV oocytes had higher concentrations of testosterone and lower progesterone/oestradiol ratios than MII oocytes, and FF of atretic oocytes contained higher testosterone concentrations than FF of MII oocytes. AMH is highly expressed in and secreted from cumulus GC of preovulatory follicles containing premature and atretic oocytes. Anti-Müllerian hormone (AMH) is produced in the female exclusively by granulosa cells. AMH has recently been shown to be one of the most important markers of ovarian reserve and it is highly associated with ovarian follicular development. This study investigates AMH expression and secretion from cumulus granulosa cells (GC) and steroidogenesis in the follicular fluids (FF) with relation to oocyte maturational stages, and fertilization capacity in large preovulatory follicles. We conducted a prospective study with 53 ovulatory women undergoing intracytoplasmic sperm injection. FF and cumulus GC from 140 large preovulatory follicles were individually obtained during oocyte retrieval. The main outcome measures were oocyte maturation, fertilization and embryo quality. FF were assayed for AMH, progesterone, 17β-oestradiol and testosterone. Cumulus GC were assayed for AMH mRNA expression. AMH mRNA expression in cumulus GC and AMH concentrations in FF of preovulatory follicles containing premature oocytes (germinal vesicle (GV) and metaphase I (MI)) were significantly higher than preovulatory follicles containing mature oocytes (MII oocytes). In addition, FF AMH concentrations of atretic oocytes were significantly higher than FF AMH of MII oocytes. No correlation was found between AMH expression and secretion for fertilization or embryo quality. FF of preovulatory MI and GV oocytes had higher levels of testosterone and lower progesterone/oestradiol ratios than MII oocytes, and FF of atretic oocytes contained higher testosterone levels than FF of MII oocytes. This study shows that AMH is highly expressed in and secreted from cumulus GC of preovulatory follicles containing premature and atretic oocytes.

Ongoing Pregnancy Rates in Women with Low and Extremely Low AMH Levels. A Multivariate Analysis of 769 Cycles

Background The ideal test for ovarian reserve should permit the identification of women who have no real chance of pregnancy with IVF treatments consequent upon an extremely reduced ovarian reserve. The aim of the current study was to evaluate pregnancy rates in patients with low AMH levels (0.2–1 ng/ml) and extremely low AMH levels (<0.2 ng/ml) and to determine the cumulative pregnancy rates following consecutive IVF treatments. Methods We conducted an historical cohort analysis at a tertiary medical center. Serum AMH levels were measured at initial clinic visit and prior to all following treatment cycles in 181 women (769 cycles) with an initial AMH level ≤1 ng/ml, undergoing IVF-ICSI. Main outcome measures were laboratory outcomes and pregnancy rates. Results Seventy patients undergoing 249 cycles had extremely low AMH levels (≤0.2 ng/ml), whereas 111 patients undergoing 520 cycles had low AMH levels (0.21–1.0 ng/ml). Number of oocytes retrieved per cycle, fertilized oocytes and number of transferred embryos were significantly lower in the extremely low AMH levels group compared to the low AMH levels (P<0.003). Crude ongoing pregnancy rates were 4.4% for both groups of patients. Among 48 cycles of women aged ≥42 with AMH levels of ≤0.2 ng/ml no pregnancies were observed. But, in patients with AMH levels of 0.2–1.0 ng/ml, 3 ongoing pregnancies out of 192 cycles (1.6%) were observed. However, in a multivariate regression analysis adjusted for age and cycle characteristics, no significant differences in ongoing pregnancy rates per cycle between the two groups were evident. Cumulative pregnancy rates of 20% were observed following five cycles, for both groups of patients. Conclusions Patients with extremely low AMH measurements have reasonable and similar pregnancy rates as patients with low AMH. Therefore, AMH should not be used as the criterion to exclude couples from performing additional IVF treatments.

Does controlled ovarian stimulation prior to chemotherapy increase primordial follicle loss and diminish ovarian reserve? An animal study

BACKGROUND Storage of embryos for fertility preservation before chemotherapy is widely practiced. For multiple oocyte collection, the ovaries are hyperstimulated with gonadotrophins that significantly alter ovarian physiology. The effects of ovarian stimulation prior to chemotherapy on future ovarian reserve were investigated in an animal model. METHODS Cyclophosphamide (Cy) in doses of 0, 50 or 100 mg/kg was administered to 38 adult mice (control, unstimulated). A second group of 12 mice were superovulated with equine chorionic gonadotrophin (eCG, 10 IU on Day 0) before Cy administration; hCG (10 IU) was administered (Day 2) followed by 0, 50 or 100 mg/kg Cy (Day 4). In both groups ovaries were removed, serially sectioned (7-day post-Cy), primordial follicles were counted and differences between groups evaluated. RESULTS Follicle number dropped from 469 +/- 24 (mean +/- SE) to 307 +/- 27 and 234 +/- 19 with 50 or 100 mg/kg Cy, respectively (P < 0.0001). In the eCG pretreated group, follicle count dropped from 480 +/- 31 to 345 +/- 16 and 211 +/- 26 when 50 or 100 mg/kg Cy were administered (P < 0.0001). There were no significant differences in follicle count between the pretreated eCG group and controls for each chemotherapy dose. CONCLUSIONS This animal study indicates that ovarian stimulation before administration of Cy does not adversely affect ovarian reserve post-treatment. These results provide support for the safety of fertility preservation using ovarian stimulation and IVF-embryo cryopreservation procedures prior to chemotherapy.

GnRH agonist vs. hCG for triggering of ovulation--differential effects on gene expression in human granulosa cells.

Objective To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of patients triggered with GnRH agonist compared to patients triggered with hCG. Design Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real time RT-PCR. Settings Single center, case control study. Patient(s) 24 women who were treated with GnRH agonist or hCG for triggering of ovulation. Interventions GC collection. Main Outcome Measure(s) The expression of genes related to steroidogenesis and OHSS in mural GCs Results The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit), CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The expression of VEGF (0.74 vs. 1) and inhibin β B (0.38 vs 1) was lower in the GnRH analog triggered group. Conclusion Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin β B in the GnRH agonist group can explain the mechanism of early OHSS prevention.