Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

PURPOSE: Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. METHODS: Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00 h. Animals were sacrificed 24 h after surgery. RESULTS: Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. CONCLUSIONS: At 11:00 h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Transabdominal punction of follicular fluid in IVF cyclus in a patient after ovarian transposition.

OBJECTIVE: Description of punction of follicular fluid in a patient after ovarian transposition. DESIGN: Case report. SETTING: Department of Obstetrics and Gynaecology, 1st Faculty of Medicine, Charles University and the General Faculty Hospital, Prague. CASE REPORT: We present a case of IVF treatment in a patient with ovarian transposition undergoing punction of follicular fluid and difficulties during this procedure acording to transabdominal route. CONCLUSION: Transabdominal punction od follicular fluid is possible, but with technical difficulities and smaller amount of obtained oocytes. We recomend to aplicate IVF procedures prior to surgical solution.

Effects of sympathectomy on ovarian follicular development and steroid secretion.

Recently, the influence of adrenergic activity over ovarian function, and thus fertility, has begun to gain importance. Previous studies have shown that adrenergic activity through norepinephrine (NE) participates in the control of follicular development and steroidal secretion from the ovary, among other functions. To examine this phenomenon, the denervation of the gonad has been widely used to observe changes in the ovary's performance. Nevertheless, the effect of the absence of adrenergic nerves in the ovary has only been studied in short times periods. In the present work, we used guanethidine (a drug that produces an irreversible sympathectomy) during the infantile period of rats, and we observed its effects in the adult rat (6 months old). Our results indicate that ovarian NE content is recovered at 6 months old, alongside with an increase of the adrenal content of NE and a dysfunctional celiac ganglion. Together, these results suggest that the recovery of ovarian NE does not come from a neural origin. In addition, ovarian performance was impaired because the changes in follicular development and steroidal secretion are not recovered despite the recovery of ovarian NE content. In conclusion, these results suggest that the nerve-ovarian connections, which are established during infantile development, are necessary for the accurate response of the ovary to sympathetic stimulation.

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels.

BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 - P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.

Puncture and drainage of the subordinate follicles at timed artificial insemination prevents the risk of twin pregnancy in dairy cows.

The objective of this study was to determine whether fluid drainage from the subordinate follicles by ovum pickup procedures prevents the risk of twin pregnancy without reducing the fertility of the cow. Lactating dairy cows with at least two follicles over 12 mm diameter located one on each ovary and selected from synchronized groups for fixed-time insemination were assigned to a Control (n = 49) or Drainage (n = 49) group. The largest follicle was considered as the dominant follicle. Draining of all subordinate follicles (≥10 mm) was performed in the Drainage group. All drained follicles developed as a corpus luteum 7 days later. In the Control cows, the presence of two corpora lutea (55.1%) was lower (p = .003) than those in the Drainage cows (87.8%), whereas the incidence of twin pregnancies was 50% and 0% for the Control and Drainage groups, respectively (p < .001). Draining did not affect the pregnancy rate. These results indicate that puncture and drainage of the subordinate follicles at insemination may eliminate the risk of twin pregnancies and reduce the risk of subsequent pregnancy loss by increasing the incidence of additional corpora lutea.

Surgical Extraction of Viable Hen ( Gallus gallus domesticus) Follicles for In Vitro Fertilization.

Assisted reproduction techniques in birds have been developed for zootechnical purposes and have been adapted for use in conservation of wild bird species. To develop a technique for obtaining follicles in live hens, 5 Rhode Island red hens ( Gallus gallus domesticus) were anesthetized, and abdominal ultrasound was performed to confirm the presence of ovarian follicles. A left celiotomy then was performed to obtain follicles in different stages of maturation for in vitro fertilization. The follicles were located by digital exploration, then extracted by isolating each follicle with the index finger of each hand, holding it by the stigma, and then applying slight traction towards the exterior of the coelomic cavity until the follicle separated from the ovary. In total, 18 of 30 (60%) follicles obtained were suitable for in vitro fertilization, but only 3 (16%) were fertilized successfully. All birds recovered from the procedure and remained in good condition postoperatively. Perfecting assisted reproduction technique holds potential benefits for determining sex of embryos by blastomeres sexing, supporting the conservation efforts of avian species, and benefiting research areas, such as genetic and biopharmaceutical research.

Follicular flushing in natural cycle IVF does not affect the luteal phase - a prospective controlled study.

In contrast to multifollicular IVF, follicular flushing seems to increase the efficacy of monofollicular IVF treatments such as natural cycle IVF (NC-IVF). However, because follicular flushing causes loss of granulosa cells, it might negatively affect luteal phase length and endocrine function of the luteal body. A prospective cohort Phase II study was performed in 24 women undergoing NC-IVF. Women underwent a reference cycle with human chorionic gonadotrophin-induced ovulation without follicle aspiration and analysis of the length of the luteal phase and luteal concentrations of progesterone and oestradiol. In addition, they underwent a NC-IVF cycle which was performed identically but follicles were aspirated and flushed three times. The luteal phase was shorter in 29.2%, equal in 16.7% and longer in 50.0% of cases following flushing of the follicles. Overall, neither difference in luteal phase length was significant [median duration (interquartile range) in reference cycle: 13 (12; 14.5), IVF (flushing) cycle: 14 (12.5; 14.5), median difference (95% CI): 0.5 (-0.5 to 1.5)] nor median progesterone and oestradiol concentrations. In conclusion, follicular flushing in NC-IVF affects neither the length of the luteal phase nor the luteal phase concentrations of progesterone and oestradiol, questioning the need for luteal phase supplementation.

Pathological assessment of follicular loss in laparoscopic endometrioma excision: effects of cyst size and surgeon's experience.

AIM: We planned to histologically demonstrate unintentional ovarian harm caused by excision of endometriomas. METHODS: One hundred and seven patients with 135 endometriomas had laparoscopic excision of endometriomas at a tertiary health-care clinic over a 6-year period. Records were obtained retrospectively and pathological specimens were reevaluated to demonstrate follicular loss. Tissue damage was scored morphologically and compared according to surgeons' experience and endometrioma size. RESULTS: Ovarian follicles were detected in 80% of endometrioma specimens near the cyst wall. Morphological scores of ovarian follicles were not affected by the surgeon's experience or endometrioma size. CONCLUSION: Surgical excision of endometriomas causes inevitable ovarian follicular loss. As observed in our study, this loss rate could not be traced to surgical experience or ovarian size.

Ovarian synchronisation in wood bison (Bison bison athabascae) during the anovulatory season.

Two experiments were performed in wood bison during the anovulatory season to establish an effective protocol for ovarian synchronisation. In an untreated control phase, bison cows (n=19) were examined daily to establish the interval to new follicular wave emergence (4.9±0.7 days) for the purposes of comparison with the experimental treatments. In Experiment 1, bison were treated by transvaginal ultrasound-guided follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol (n=10). In Experiment 2, bison were treated by follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol +100mg, i.m., progesterone (n=10). In Experiment 1, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol-treated groups was 4.9±0.7, 1.1±0.1 and 3.1±0.4 days, respectively (P<0.05). The degree of synchrony was 2.4±0.4, 0.2±0.1 and 0.8±0.2 days, respectively (P<0.05). In Experiment 2, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol + progesterone-treated groups was 4.9±0.7, 1.2±0.2 and 3.3±0.3 days, respectively (P<0.05), and the degree of synchrony was 2.4±0.4, 0.2±0.1, and 0.8±0.2 days, respectively (P<0.05). The degree of synchrony did not differ between ablation and hormone treatment groups in either experiment, but was greater in treatment groups than in the untreated control phase. Both follicular ablation and hormone treatment shortened and decreased the variability in the interval to follicular wave emergence in bison, but wave emergence occurred earlier after follicular ablation.

Live birth following vitrification of in vitro matured oocytes derived from sibling smaller follicles at follicle selection phase in the context of in vitro fertilization.

In ovarian stimulation, a 31-year-old woman with polycystic ovary syndrome was at the risk of developing ovarian hyperstimulation syndrome, follicle aspiration was performed, and eight immature oocytes were collected from follicle fluids. After 28 h in vitro culture, six of them reached MII and were vitrified. The patient failed to conceive in her fresh in vitro fertilization cycle and next two replacement cycles. In the third replacement cycle, a successful pregnancy was obtained by vitrified-thawed oocytes. This case demonstrates that follicular aspiration during follicle selection phase has protective effects against developing ovarian hyperstimulation syndrome, and rescued immature oocytes are viable and could produce promising embryos for live birth.

Anti-Müllerian hormone as a marker of ovarian reserve in patients with ovarian malignancies who have undergone fertility-preserving surgery and chemotherapy.

With proper and careful selection of patients, fertility-preserving surgery may be feasible in patients with ovarian malignancies. However, the loss of follicles by oophorectomy and chemotherapy results in decreased ovarian reserve, which consecutively affects reproductive capacity. We evaluated postoperative levels of serum anti-Müllerian hormone (AMH) in women with ovarian malignancies to assess the impact of the fertility-preserving surgery with or without the administration of chemotherapy on ovarian reserve. In 13 patients who underwent the fertility-preserving surgery with (n = 9) or without (n = 4) the administration of chemotherapy, serum AMH levels were measured and compared with serum AMH levels measured in patients undergone cystectomy for benign ovarian tumors as a control. We found that the mean AMH level in the treatment group measured 0.9 ng/mL, which was significantly lower than that measured in the control group (4.70 ± 3.77 ng/mL). The possibility of decreased ovarian reserve occurring in patients with ovarian malignancies following treatment with fertility-preserving surgery with or without the administration of chemotherapy should be considered for fertility planning.

Adaptation of a trap door technique for the recovery of ovarian cortical biopsies from Cebus apella (capuchin monkey).

There is a paucity of efficient cryopreservation protocols for primordial follicles enclosed in the ovarian tissue from non-human primates (NHP), in special New World primates. Our objective was to establish an optimal procedure for the recovery of ovarian biopsies from capuchin monkeys. To this end, we adapted a trap door biopsy method. Follicular density and quality of the biopsies were evaluated and ultrasound analysis was performed before and continuously after surgery to assess ovarian structure. Ovarian tissue biopsies recovered by the trap door technique allowed the successful harvesting of primordial follicles from capuchin monkeys, and no complication was recorded. The female cycle was not affected by surgery and no adherence was found thereafter. In conclusion, the adaptation of a trap door biopsy method is a safe procedure and allows recovery of healthy primordial follicles.

Retrieval and in vitro maturation of human oocytes from ovaries removed during surgery for endometrial carcinoma: a novel strategy for human oocyte research.

PURPOSE: To collect human oocytes from ovaries removed as part of surgical treatment for endometrial carcinoma, and to induce in vitro maturation of such oocytes to obtain material for research on human ovarian aging. DESIGN: Prospective clinical study. SETTING: University Hospital. PATIENTS: Eight patients aged 35-44 years with a preoperative diagnosis of Stage I endometrial cancer agreed to participate in this project. INTERVENTIONS: Surgically removed ovaries were punctured; oocytes were collected from follicular fluid and matured in vitro. Immunofluorescent detection of microtubules and DNA labeling were performed after in vitro maturation. MAIN OUTCOME MEASURES: Number of oocytes collected and their in vitro maturation stage. RESULTS: In total, 87 oocytes were collected, 11 of which had completed metaphase II. Of the oocytes collected, 75 % were from three patients in their 30s, while the remaining 25 % were from five patients in their 40s. Several stages of oocytes were collected and the detection of microtubule arrangement and chromatin in various stages using fluorescence was possible. CONCLUSION: Material for research on human ovarian aging can be obtained from ovaries removed during surgery for endometrial cancer.

Attempted application of bioengineered/biosynthetic supporting matrices with phosphatidylinositol-trisphosphate-enhancing substances to organ culture of human primordial follicles.

PURPOSE: To improve human primordial follicle culture. METHODS: Thin or thick ovarian slices were cultured on alginate scaffolds or in PEG-fibrinogen hydrogels with or without bpV (pic), which prevents the conversion of phosphatidylinositol-trisphosphate (PIP3) to phosphatidylinositol-bisphosphate (PIP2) or 740Y-P which converts PIP2 to PIP3. Follicular growth was evaluated by follicular counts, Ki67 immunohistochemistry, and 17ß-estradiol (E2) levels. RESULTS: BpV (pic) had a destructive effect on cultured follicles. Thawed-uncultured samples had more primordial follicles than samples cultured in basic medium and fewer developing follicles than samples cultured in PEG-fibrinogen hydrogels with 740Y-P. There were more atretic follicles in samples cultured on alginate scaffolds than in PEG-fibrinogen hydrogels, and in samples cultured in PEG-fibrinogen hydrogels with 740Y-P than in PEG-fibrinogen hydrogels with basic medium. Ki67 staining was higher in PEG-fibrinogen hydrogels than on alginate scaffolds. E2 levels were higher in thick than in thin slices. CONCLUSIONS: PEG-fibrinogen hydrogels appear to have an advantage over alginate scaffolds for culturing human primordial follicles. Folliculogenesis is not increased in the presence of substances that enhance PIP3 production or with thin rather than thick sectioning.

Role of follicular estradiol-17beta in timing of luteolysis in heifers.

The hypothesis was tested that estradiol (E2) from the ovarian follicles controls time of luteolysis. Time of luteolysis was evaluated by multiple measures of corpus luteum (CL) structure (area, volume) and function (progesterone [P4], luteal blood flow). The hypothesis for experiment 1 was that repeated ablation of follicles would reduce circulating E2 and delay luteolysis. Heifers were randomly assigned on Day 9 (Day 0 = ovulation) to three groups. All follicles >or=4 mm were ablated on Day 9 (group FA9; n = 6); Days 9-15 (group FA15; n = 6); or Days 9-21 (group FA21; n = 7). As expected, follicular ablation delayed (P < 0.001) the rise in circulating E2 and peak E2 concentrations (FA9, Day 17.6 +/- 0.7; FA15, Day 20.3 +/- 0.3; FA21, Day 24.9 +/- 0.3). Luteolysis (based on each measure) was delayed (P < 0.005) by repeated ablation of follicles, with earlier luteolysis (based on P4 decrease) in FA9 (Day 15.2 +/- 0.8) than FA15 (Day 16.5 +/- 0.4), and a further delay in FA21 (Day 18.3 +/- 0.5). The hypothesis of experiment 2 was that exogenous treatment with E2 would stimulate prostaglandin F(2alpha) (PGF) secretion and prevent the delay in luteolysis associated with follicular ablations. Follicles >or=4 mm were ablated from Day 9 to Day 17 (n = 15). Heifers were treated on Days 13 and 15 with 1.0 mg of estradiol benzoate (FAE2; n = 7) or vehicle (FAV; n = 8). Treatment with E2 induced PGF secretion (detected by PGF metabolite) and induced earlier (P < 0.02) luteolysis in FAE2 than in FAV, whether determined by circulating P4 or by area, volume, or blood flow of CL. In summary, ablation of follicles (>or=4 mm) delayed and treatment with E2 hastened luteolysis in heifers with ablated follicles. Thus, these results are consistent with an essential role for follicle E2 in timing of luteolysis.

Development of one vs multiple ovulatory follicles and associated systemic hormone concentrations in mares.

Ablation of follicles > or = 6 mm in diameter and treatment with PGF2alpha 10 days after ovulation were used to induce the development of ovulatory waves. Comparisons were made between induced waves with one (33 waves, 72%) and multiple (13 waves, 28%) ovulatory follicles. Diameter deviation was defined as the separation of follicles into dominant and subordinate categories. Multiple ovulatory follicles were preceded by more (p < 0.001) follicles > or = 20 mm at the beginning of deviation, higher LH preceding deviation (approached significance, p < 0.08), lower (p < 0.05) concentrations of FSH on the day of deviation and thereafter, and higher (p < 0.0003) oestradiol by 2 days after deviation. During the peri-ovulatory period, systemic hormone concentrations for waves with multiple ovulations involved higher oestradiol before ovulation (approached significance, p < 0.07), lower FSH (p < 0.04) before and after ovulation, and both higher progesterone (p < 0.05) and lower LH (p < 0.05) beginning the day after ovulation. Results indicated that by the beginning of deviation there were more follicles > or = 20 mm and subsequently greater oestradiol production in waves that led to the development of multiple ovulatory follicles, and during the peri-ovulatory period differences between one and multiple ovulations were consistent with the negative effects of the ovarian hormones on the gonadotropins.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares.

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.

Maturational costs of reproduction due to clutch size and ontogenetic conflict as revealed in the invisible fraction.

Reproduction is thought to entail costs, but assessing survival costs associated with maturation as organisms express reproductive genes for the first time is problematic because many will die prior to expressing a phenotype. We quantified selection acting on this invisible fraction by measuring selection on predicted breeding values for clutch size estimated from a multigenerational pedigree of side-blotched lizards in which clutch size was heritable (h2=0.25+/-0.04). The survival effects of predicted breeding values for clutch size during maturation, however, differed between males and females indicating ontogenetic conflict. Increased mortality during maturation was associated with lower predicted breeding values for clutch size for females, but higher predicted breeding values for males who do not produce a clutch. Genetic correlations between clutch size and male and female survival were consistent with calculated selection differentials. Experimental yolk removal did not affect progeny survival during maturation, indicating that selection on maturing progeny was not due to confounding yolk-volume maternal effects, and hormone manipulations confirmed clutch size as the target of viability selection during maturation. Such episodes of selection prior to phenotypic expression of the trait will have important consequences for the evolution of reproductive investment.

Follicular aspiration versus coasting for ovarian hyper-stimulation syndrome prevention.

OBJECTIVE: To compare follicular reduction prior to human chorionic gonadotropin (HCG) trigger and coasting in terms of ovarian hyper-stimulation syndrome (OHSS) reduction, pregnancy, and cancellation rates in in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) cycles. METHODS:  This study was designed as a prospective study. The setting was the IVF unit at King Abdulaziz Medical City, Riyadh, Kingdom of Saudi Arabia. A total of 39 patients undergoing IVF/ICSI cycles, who were at risk of OHSS, 20 were put into a coasting group and 19 had follicular reduction instead. This occurred between October 2010 and January 2011. Our main outcome was OHSS reduction. RESULTS: Six (30%) women developed OHSS in the coasting group and 2 (10.5%) women developed OHSS in the follicular group (p-value=0.235). The pregnancy rates in the cycles were similar for both groups: 4/20 (20%) in the coasting group and 3/19 (15.8%) in the follicular group (p-value=1.000). The cancellation rate of the cycles was similar for both groups, 6/20 (30%) in the coasting group and 1/19 (5.3%) in the follicular group (p-value=0.09). The median number of punctured follicles was significantly lower in the follicular group (16 follicles, interquartile range (IQR)=21-12) compared to the coasting group (29 follicles, IQR=37.8-19.8, p-value=0.001). The retrieved, fertilized, and cleaved oocytes, as well as the number of embryos transferred, were similar amongst both groups. CONCLUSION: There was no difference between follicular reduction prior to HCG and coasting, in terms of OHSS reduction, pregnancy, and cancellation rates in both the IVF and ICSI cycles.

Ovotesticular Disorder of Sex Development Presenting as an Acute Scrotum.

Hermaphroditism is known as ovotesticular disorder of sex development. A 14-year-old boy was admitted with right acute scrotum. Exploration revealed tunica rupture and hematoma, with no viable tissue. After 1 month, he was admitted again with left hemiscrotal pain. Microscopic examination of the left gonad demonstrated foci of hemorrhagic cysts, primordial follicles, and regions of seminiferous tubules. We preserved a testicular tissue and the ovarian part was extracted completely. Long-term follow-up with his hormonal profile is reported. This is a case of ovotesticular disorder presented with acute scrotum and we also tried to reduce long-term hormone therapy, with preservation of testicular part.