Search

Robotic surgery is a conceptual fusion of the conventional open surgery and the minimally invasive laparoscopic surgery. We reviewed the current role of robotic-assisted laparoscopy in the field of reproductive surgery by a literature search in PubMed database. We analyzed the reported advantages and limitations of the use of robotics in reproductive surgeries like myomectomy, tubal reanastomosis, endometriosis, ovarian tissue cryopreservation, and ovarian transposition. Overall, robotic assistance in reproductive surgery resulted in decreased blood loss, less post-operative pain, shorter hospital stay, and faster convalescence, whereas reproductive outcomes were similar to open/laparoscopic approaches. The main drawbacks of robotic surgery were higher cost and longer operating times. It is as safe and effective as the conventional laparoscopy and represents a reasonable alternate to abdominal approach. Procedures that are technically challenging with the conventional laparoscopy can be performed with robotic assistance. It has advantages of improved visualization and Endowrist™ movements allowing precise suturing. This helps to overcome the limitations of laparoscopy, especially in complicated procedures, and may shorten the steep learning curve in minimal invasive surgery. Randomized controlled trials looking at both short- and long-term outcomes are warranted to strengthen the role of robotic surgery in the field of reproductive surgery.

Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.—Martellini, J. A., Cole, A. C., Venkataraman, N., Quinn, G. A., Svoboda, P., Gangrade, B. K., Pohl, J., Sørensen, O. E., Cole, A. M. Cationic polypeptides contribute to the anti-HIV-1 activity of human seminal plasma.

The inability of healthcare provider to conclusively identify the cause of infertility in couples leads to the designation of unexplained infertility. It is likely that abnormalities leading to infertility exists in couples but not detected by the current methods and the state of technology. Normal semen parameters in men reduce but do not completely eliminate the probability of male infertility. The wide range in World Health Organization (WHO) reference values of semen parameters, repeated revisions of normal reference range over the years, significant variation in interlaboratory assessment of sperm characteristics, and use of different analytical criteria (WHO versus Krueger’s Strict morphology) further complicates the clinical assessment of normal semen parameters. High incidence of chromosomal aberrations and/or high percentage of DNA damage in sperm, oxidative stress, exposure of men to environmental pollutants/toxins, etc. may be likely factors that can be the suspected cause of unexplained infertility. Abnormal expression of certain sperm proteins may play a role in failure of fertilization resulting in unexplained male infertility at clinical level. Unexplained male infertility is strongly suspected in couples if the female partner presents with the history of successful pregnancy in not too distant past and subsequent comprehensive diagnostic tests fail to lead to a confirmed infertility diagnosis.

The successful outcome following in vitro fertilization and embryo transfer (IVF-ET) depends on a large number of variables ranging from maternal age, cause of infertility, number and maturity of oocytes, sperm characteristics, embryo quality, and ultimately the quality of embryo transfer. In the order of significance the two parameters that influence the outcome of IVF most are the age of the female patient (or the egg donor) followed by the quality of the embryo(s) transferred. It is well established that the morphological characteristics and the quality of the embryos are largely dependent on the oocyte (Hum Reprod 12:1750–5, 1997; Hum Reprod 12:1267–70, 1997; Fertil Steril 72:240–4, 1999; J Assist Reprod Genet 16:287–93, 1999). A combination of sperm characteristics such as concentration, morphology, and motility offers some predictive value as it relates to the fertilization of oocytes in vitro; however, their importance in predicting embryo morphology is controversial. In fact the contribution of sperm to the process of reproduction can be divided into three steps: (1) transfer of paternal genome to the oocyte; (2) activation of the oocyte; and (3) transfer of centriole/centrosome to the oocyte to assist in the organization of mitotic spindle. It is only logical to assume that any abnormality or defect in the spermatozoon fertilizing the oocyte will be reflected in the embryonic development and pregnancy outcome following transfer of the embryo. The term “embryo quality” generally refers to the morphological assessment of the embryo; however, in terms of implantation potential the embryo quality suitably includes criteria such as the rate of embryonic cleavage and its genetic (chromosomal) status. Several publications have analyzed the relationship between sperm morphology and implantation and/or pregnancy outcome with little or no mention of embryo quality. Since successful implantation and clinical pregnancies are indirect indication of potentially good quality embryos, these studies have been included in this review.

Retrieval and cryopreservation of testicular sperm has become a cornerstone of infertility treatment in azoospermic men. In obstructive azoospermia, sperm may be retrieved from testis in almost 100% patients and can be frozen in multiple aliquots to avoid repeated surgeries. In approximately 50% of patients with nonobstructive azoospermia, enough sperm may be retrieved for IVF–intracytoplasmic sperm injection (ICSI). Any excess sperm remaining after ICSI can be frozen for future IVF attempts. Frozen testicular sperm are as effective in achieving successful pregnancies as freshly isolated testicular or ejaculated sperm. Treatment with motility-enhancing agents, such as pentoxifylline, induces motility in frozen-thawed testicular sperm and facilitates selection of viable sperm for ICSI. Special techniques, such as cryopreservation of single sperm within evacuated hamster zona or on a cryoloop, allow storage of extremely poor sperm samples. In USA, at this time, there is no legislative directive with respect to the posthumous procurement of sperm.

Sterile culture dishes Polystyrene conical tubes Sterile glass pipettes Syringes (50 mL) Syringe (3 mL) with 21 gauze needle Syringe filter (Nalgene, 0.22 μm) Microscope glass slides

The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. The techniques for the retrieval of spermatozoa vary from relatively simple percutaneous sperm aspiration to open excision (testicular biopsy) and the more invasive Micro-TESE. The probability of retrieving spermatozoa can be as high as 100% in men with obstructive azoospermia (congenital bilateral absence of the vas deferens, status post-vasectomy). However, in non-obstructive azoospermia, successful sperm retrieval has been reported in 10-100% of cases by various investigators. The surgical retrieval and cryopreservation of sperm, especially in men with non-obstructive azoospermia, to some extent ensures the availability of sperm at the time of intracytoplasmic sperm injection. In addition, this strategy can avoid unnecessary ovarian stimulation in those patients intending to undergo in vitro fertilization-intracytoplasmic sperm injection with freshly retrieved testicular sperm when an absolute absence of sperm in the testis is identified. Several different methods for the cryopreservation of testicular and epididymal sperm are available. The choice of the container or carrier may be an important consideration and should take into account the number or concentration of the sperm in the final preparation. When the number of sperm in a testicular biopsy sample is extremely low (e.g., 1-20 total sperm available), the use of an evacuated zona pellucida to store the cryopreserved sperm has been shown to be an effective approach.

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase ( PAP) have been reported to form amyloid fibrils called "SEVI'' and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

We have investigated the effects of purified alpha-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly (P0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Porcine granulosa cells isolated from small (1–3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-β (TGFβ) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGFβ to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGFβ in the theca cell conditioned medium was quantitatively estimated by generating a TGFβ-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGFβ-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGFβ-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent ...

We sought to provide a cost-beneficial approach to in vitro fertilization for infertile patients who could not afford the standard treatment with in vitro fertilization and to determine the optimal level of minimal ovarian stimulation to achieve acceptable pregnancy rates.We performed a retrospective cohort study of 216 patients who underwent "minimal stimulation" in vitro fertilization between January 1994 and December 1998. During the first half of this study, various minimal ovarian stimulation protocols were performed in our private, free-standing center for in vitro fertilization. More recently, more ovarian stimulation, including a 4-day protocol featuring gonadotropin-releasing hormone agonist flare (ultrashort flare), was used. Clinical pregnancy outcome, multiple gestation, complications, and maternal age were compared between the first and second halves of this study.The average ages of patients in the first half (phase 1) and the second half (phase 2) were similar, 32.4 +/- 0.3 versus 32.6 +/- 0.3 years, respectively. An average of 3.5 oocytes per retrieval was obtained in phase 1 versus 5.9 oocytes in phase 2. Failure to retrieve oocytes occurred in 3% of all cases. The mean number of embryos transferred per patient was 2.0 in phase 1 versus 2.4 in phase 2. In phase 1, 16.1% of patients failed to have viable embryos for transfer, in comparison with 9.7% in phase 2. The overall clinical pregnancy rate per retrieval was 16.9% in phase 1 versus 36. 6% in phase 2. Multiple gestation occurred in 5.0% of clinical pregnancies in phase 1 but increased to 33% in phase 2, with 9 sets of twins and 6 sets of triplets. The implantation rate was 9.3% for phase 1 versus 23.3% for phase 2. The clinical pregnancy rates per retrieval for phase 2 patients were 41.6% in womenor =34 years old and 25.6% for thoseor =35 years old. No case of ovarian hyperstimulation syndrome was noted.Minimal ovarian stimulation in the setting of in vitro fertilization offers a cost-beneficial alternative to standard treatment with in vitro fertilization in infertile patients who are35 years old and in women40 years old who have adequate oocyte reserve. More stimulation improves outcome. Minimalstimulation in vitro fertilization provides an alternative for those patients who cannot afford standard in vitro fertilization or who are concerned with exposure to high dosages of fertility medications.

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.

Transforming growth factor-beta (TGF-β), a multifunctional polypeptide growth factor, is produced by follicular cells in the ovary. However, there is little information indicating that TGF-β is produced in the post-ovulatory follicle, i.e. the corpus luteum. In the present communication we present evidence that bovine luteal cells secrete large amounts of TGF-β when maintained in moderate-term monolayer culture. Using TGF-β1 and TGF-β2 subtype-specific antibodies to neutralize the bioactivity it was found that 80–90% TGF-β activity in luteal cell conditioned medium (LCCM) is due to TGF-β1, whereas ≤ 10% TGF-β activity in LCCM is due to TGF-β2. TGF-β subtype nonspecific antibodies effectively and completely neutralized all TGF-β activity in LCCM. The ratio of TGF-β1:TGF-β2 as estimated on the basis of neutralization studies was supported by visual observation of TGF-β1 and TGF-β2 protein bands on Western blotting. Using a modified and rapid mink lung epithelial cell bioassay and authentic TGF-β to generate standard curves, the amount of TGF-β secreted by luteal cells in vitro was quantitated. The concentration of luteal cell secreted proteins in the medium increased with time during 7 days of culture. Likewise, the TGF-β concentration in LCCM increased during 7 days. To study the effect of duration of culture on the rate of TGF-β secretion by luteal cells, conditioned medium was collected at 24 h intervals and replaced with fresh nutrient medium. The rate of TGF-β secretion increased gradually on days 2, 3 and 4 of culture and declined slowly thereafter. Interestingly, measurement of the rate of total protein secretion into the conditioned medium at 24 h intervals showed a steady decline with the duration of culture. The significant decrease in protein secretion by luteal cells during the latter part of prolonged culture resulted in an increase in TGF-β secretion per unit total protein secretion (i.e. ng TGF-β/μg protein). These studies for the first time demonstrate that bovine luteal cells secrete high amounts of latent TGF-β1 and relatively little TGF-β2. It is suggested that luteal cell secreted TGF-β may play a role as an autocrine or paracrine regulator of luteal function and may modify gonadotropin-dependent ovarian follicular functions.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.