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.

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.

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.

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.

Ultrasound-guided follicle aspiration at time of laparotomy in a patient with Mayer-Rokitansky-Küster-Hauser syndrome.

OBJECTIVE: To demonstrate a unique case of direct ultrasound-guided follicle aspiration at time of a laparotomy in a patient with Mayer-Rokitansky-Küster-Hauser Syndrome. DESIGN: Video presentation. SETTING: Academic fertility center PATIENT(S): A 32-year-old gravida 0 with Mayer-Rokitansky-Küster-Hauser Syndrome and bilateral ovarian masses presented as a fertility preservation consult from gynecologic oncology due to the possibility of bilateral oophorectomy. Due to the appearance and size of the left ovary oncology planned to perform an exploratory laparotomy and left oophorectomy, with possible right oophorectomy. The patient and her partner desired embryo cryopreservation with plans for future use in a gestational carrier. She had previously undergone vaginal dilator therapy, however her ovaries were inaccessible transvaginally due to their cephalad location and small caliber of the vaginal pouch. The plan was made to proceed with controlled ovarian stimulation and concurrent ultrasound-guided follicle aspiration of the right ovary at the time of laparotomy following left oophorectomy. INTERVENTION(S): Ultrasound-guided follicle aspiration in vivo at time of laparotomy. MAIN OUTCOME MEASURE(S): Successful controlled ovarian stimulation, oocyte retrieval and embryo cryopreservation. RESULT(S): The patient underwent a long agonist protocol and received a total of 2,525 units of gonadotropin with a peak estradiol of 3,264 pg/ml. She required a total of 9 days of stimulation. The normal right ovary responded as expected, and the left ovary remained unchanged. Following laparotomy and left oophorectomy, direct application of the transvaginal ultrasound probe was used to aspirate all visible follicles on the right side in vivo. Twenty-four oocytes were retrieved, 15 were mature and 5 blastocysts were cryopreserved. Final pathology of left ovary returned as serous cystadenoma. The right ovary was examined by gynecologic oncology prior to and following retrieval and was thought to be normal and remained in situ. CONCLUSION(S): Although the approach described here is not feasible in most cases, this video demonstrates a unique and successful fertility preservation technique by direct ultrasound-guided follicle aspiration in vivo at the time of laparotomy in a Mayer-Rokitansky-Küster-Hauser Syndrome patient and to our knowledge, is the first description of its kind. This retrieval would have otherwise been limited by lack of access transvaginally and limited visualization transabdominally. This combined approach should be considered in future patients with müllerian anomalies and similar complicating factors necessitating laparotomy.

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 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.

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.

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.

Single-incision, prefemoral bilateral oophorosalpingectomy without coelioscopy in an Indian star tortoise (Geochelone elegans) with follicular stasis.

A female Indian star tortoise (Geochelone elegans) was evaluated for anorexia and suspected coelomic masses. Clinical examination indicated follicular stasis. The tortoise was anesthetized and placed in dorsal left lateral recumbency for a right prefemoral approach. The ovaries and oviducts were exposed through the right prefemoral incision. The ovarian vasculature, the mesovarium, the oviduct, mesosalpinx and associated vasculature were ligated and transected. Bilateral oophorosalpingectomy was performed through the incision, without coelioscopy. Since then, the same procedure has been performed in five other tortoises, and all recovered well. These results suggest that bilateral oophorosalpingectomy, performed through a prefemoral incision, without coelioscopy, is a safe and practical approach for treating follicular stasis in tortoises.

Restricting the induction of NGF in ovarian stroma engenders selective follicular activation through the mTOR signaling pathway.

In mammalian ovaries, primordial follicles remain in a quiescent state until activation by the surrounding microenvironment. Ovarian intervention, for example, ovarian cystectomy, ovarian wedge resection or laser drilling therapies for polycystic ovarian syndrome, has long been reported to change follicular development by an unknown mechanism(s). Herein, we established a murine model with partial ovarian resection of one ovary unilaterally, with the contralateral ovary undamaged. We found the injury accelerated follicular activation and development through the mTORC1 signaling pathway. Moreover, the stimulation of primordial follicles was restricted near the incision site where the mTORC1 pathway showed sequential activation beginning at the interstitial cells and proceeding to the primordial follicles. Total and polysome-associated RNA-seq revealed the increase of the nerve growth factor (NGF) family member, in both two fractions and immunostaining showed the restricted induction of NGF near the incision site. In cultured newborn ovaries, NGF demonstrated increase of follicular activation, and moreover, the NGF inhibitor K252a effectively blocked activation of primordial follicles stimulated by the surgery. We liken ovulation in mammals to minor tissue trauma, which happens naturally and cyclically in the body. As the increase in NGF accompanied the accumulation of activated primordial follicles after ovulation, our study may represent a common mechanism for selective follicular activation induced by a localized increase in NGF in interstitial cells and mediated via the mTOR signaling pathway. In addition, the NGF inhibitor K252a and the mTOR inhibitor rapamycin constitute good candidates for protecting follicular reserve against over exhaustion after ovarian surgery.

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.

An intraovarian mechanism that enhances the effect of an FSH surge on recovery of subordinate follicles in heifers.

The effect of the future dominant follicle (DF), corpus luteum (CL), and side (left ovary [LO] and right ovary [RO]) on FSH-induced recovery (increase in diameter) of regressing subordinate follicles was studied in heifers. The DF of wave 2 and the largest subordinate follicle remained intact (controls, n = 14 heifers) or were ablated (n = 14 heifers) on a mean of 13 d postovulation when the DF was ∼10 mm (hour 0). Concentration of FSH (P < 0.0004) and diameter of subordinate follicles (P < 0.0002) decreased between hours -48 to 0 combined for the control and ablation groups. Thereafter, follicle diameter continued to decrease in the controls. Concentration of FSH increased (P < 0.05) and diameter of subordinates began to increase at hour 12 in the ablation group. Follicle-stimulating hormone increased to hour 24 and then returned to the hour 0 concentration by hour 72, completing the induced FSH surge. Concentration of LH began to increase at hour 0 in each group and at a similar rate between groups. Follicle recovery in the ablation group was compared among 8 subgroups as defined by the 2 sides and 4 intraovarian patterns (DF-CL pattern, both structures in same ovary; DF pattern, DF alone; CL pattern, CL alone; and devoid pattern, both structures absent). Follicle diameter increased (P < 0.05) between hours 24 and 48, and diameter at hours 24, 48, 72, and 96 involved a 3-way interaction (P < 0.0001) of pattern, side, and hour. The interaction was similar when diameter of the DF that originated from a recovered subordinate was either included or excluded in the analysis. Diameter of subordinate follicles in the ablation group at hour 96 was greater (P < 0.05) in the DF-CL/RO and DF/RO subgroups than that in the devoid/LO, devoid/RO, and CL/LO subgroups. The DF-CL/LO and CL/RO subgroups were intermediate. For follicles that decreased in diameter before hour 0, a greater (P < 0.05) percentage increased after hour 0 when the ovary contained a DF and was in the RO (DF-CL/RO and DF/RO subgroups) than for the remaining subgroups even after excluding the DF that originated from a subordinate. Results supported the hypotheses that (1) an induced FSH surge can stimulate the recovery of regressing subordinate follicles and (2) recovery of regressing subordinate follicles by FSH involves an intraovarian mechanism. Our interpretation is that the intraovarian mechanism that enhances the stimulatory effect of FSH on recovery of subordinate follicles was effective only in RO and only when it contained a DF.

Influence of transvaginal ultrasound-guided follicular punctures in the mare on heart rate, respiratory rate, facial expression changes, and salivary cortisol as pain scoring.

Transvaginal ultrasound-guided follicular punctures are widely used in the mare for diagnosis, research, and commercial applications. The objective of our study was to determine their influence on pain, stress, and well-being in the mare, by evaluating heart rate, breath rate, facial expression changes, and salivary cortisol before, during, and after puncture. For this experiment, 21 pony mares were used. Transvaginal ultrasound-guided aspirations were performed on 11 mares. After injections for sedation, analgesia, and antispasmodia, the follicles from both ovaries were aspirated with a needle introduced through the vagina wall into the ovary. In the control group, 10 mares underwent similar treatments and injections, but no follicular aspiration. Along the session, heart rate and breath rate were evaluated by a trained veterinarian, ears position, eyelid closure, and contraction of facial muscles were evaluated, and salivary samples were taken for evaluation of cortisol concentration. A significant relaxation was observed after sedative injection in the punctured and control mares, according to ear position, eyelid closure, and contraction of facial muscles, but no difference between punctured and control animals was recorded. No significant modification of salivary cortisol concentration during puncture and no difference between punctured and control mares at any time were observed. No significant modification of the breath rate was observed along the procedure for the punctured and the control mares. Heart rate increased significantly but transiently when the needle was introduced in the ovary and was significantly higher at that time for the punctured mares than that for control mares. None of the other investigated parameters were affected at that time, suggesting discomfort is minimal and transient. Improving analgesia, e.g., through a multimodal approach, during that possibly more sensitive step could be recommended. The evaluation of facial expression changes and heart rate is easy-to-use and accurate tools to evaluate pain and well-being of the mare.

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.

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.

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.

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.

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.