E.L.A. Motta

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification

OBJECTIVE To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. DESIGN Prospective randomized. SETTING Academically affiliated, private fertility center. PATIENT(S) Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. INTERVENTION(S) Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. MAIN OUTCOME MEASURE(S) Oocyte survival, fertilization, embryo development, and clinical pregnancy. RESULT(S) Patient use has resulted in 30 thaws and 48 warmings. Womens age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. CONCLUSION(S) Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy.

Extensive Excision of Deep Infiltrative Endometriosis before In Vitro Fertilization Significantly Improves Pregnancy Rates

STUDY OBJECTIVE We sought to compare the outcomes of in vitro fertilization (IVF) treatments in women with infertility-associated deep infiltrative endometriosis (DIE) who underwent extensive laparoscopic excision of endometriosis before IVF with those who underwent IVF only. DESIGN Prospective cohort study. SETTING Infertility clinic and private hospital in São Paulo, Brazil. PATIENTS A total of 179 infertile patients younger than 38 years had symptoms and/or signs of endometriosis and sonographic images suggestive of DIE. INTERVENTIONS After thorough counseling, 179 women were invited to participate in a prospective cohort study with 2 treatment options: IVF without undergoing laparoscopic surgery (group A, n = 105) and extensive laparoscopic excision of DIE before IVF (group B, n = 64). Ten women were lost to follow-up. The IVF outcomes were compared between the 2 groups. MEASUREMENTS AND MAIN RESULTS In group B, patients had 5 +/- 2 (mean +/- SD) DIE lesions excised during laparoscopy. Patient characteristics in groups A and B, respectively, were: age (32 +/- 3 vs 32 +/- 3 years, p = .94), infertility duration (29 +/- 20 vs 27 +/- 17 months, p = .45), day-3 serum follicle-stimulating hormone levels (5.6 +/- 2.5 vs 5.9 +/- 2.5 IU/L, p = .50), and previous IVF attempts (1 +/- 1 vs 2 +/- 1, p = .01). The IVF outcomes differed between groups A and B, respectively, with regard to total dose of recombinant follicle-stimulating hormone required to accomplish ovulation induction (2380 +/- 911 vs 2542 +/- 1012 IU, p = .01), number of oocytes retrieved (10 +/- 5 vs 9 +/- 5, p = .04), and pregnancy rates (24% vs 41%, p = .004), but not number of embryos transferred (3 +/- 1 vs 3 +/- 1, p = 1). The odds ratio of achieving a pregnancy were 2.45 times greater in group B than in group A. CONCLUSION Extensive laparoscopic excision of DIE significantly improved IVF pregnancy rates of women with infertility-associated DIE.

The role of the Hoxa10/HOXA10 gene in the etiology of endometriosis and its related infertility: a review

PurposeEndometriosis and its associated infertility have been the object of continuous research for over a century. To understand the molecular mechanisms underlying the disease, it has become necessary to determine the aspects of its etiology that are not explained by the retrograde menstruation theory. This could in turn elucidate how various clinical and surgical treatments might affect the evolution and remission of the disease.MethodsThis review is focused on the most recent clinical and laboratory findings regarding the association of HOXA10 with endometriosis and infertility.ResultThe homebox (Hox/HOX) proteins are highly conserved transcription factors that determine segmental body identities in multiple species, including humans. Hoxa10/HOXA10 is directly involved in the embryogenesis of the uterus and embryo implantation via regulation of downstream genes. Cyclical endometrial expression of Hoxa10/HOXA10, with a peak of expression occurring during the window of implantation, is observed in the adult in response to estrogen and progesterone. Women with endometriosis do not demonstrate the expected mid-luteal rise of HOXA10 expression, which might partially explain the infertility observed in many of these patients. Recent studies also demonstrated HOXA10 expression in endometriotic foci outside the Müllerian tract.ConclusionsMultiple lines of evidence suggest that the actions of the homeobox A10 (Hoxa10/HOXA10) gene could account for some aspects of endometriosis.

High implantation and pregnancy rates with transfer of human blastocysts developed in preimplantation stage one and blastocyst media

OBJECTIVE To evaluate the proficiency of preimplantation stage one (P1) and blastocyst media in supporting human blastocyst development and to document implantation and clinical pregnancy rates from the transfer of the normally developed blastocysts. DESIGN Retrospective clinical study. SETTING Private IVF unit of a university-affiliated center. PATIENT(S) Twenty-eight women aged 33.7 +/- 2.9 years who underwent IVF treatment for infertility. INTERVENTION(S) Bipronucleate oocytes obtained from IVF and intracytoplasmic sperm injection were cultured in vitro with P1 and blastocyst media for 96-120 hours. One to three embryos were transferred (2.1 +/- 0.2 for the patients who became pregnant and 1.5 +/- 0.3 for those who did not become pregnant). MAIN OUTCOME MEASURE(S) Total number and percentage of developed blastocysts, frequency of blastocysts of grades A and B, and implantation and pregnancy rates. RESULT(S) From 431 oocytes retrieved, 269 bipronucleate oocytes were cultured, producing 81 blastocysts that resulted in the transfer of 54 embryos in 27 procedures. Blastocysts developed in 39.7% +/- 5.5% of the pregnant group and 30.2% +/- 4.5% of the nonpregnant group. From 15 (15/27 = 55.6%) clinical pregnancies, 18 (18/54 = 33.3%) gestational sacs were visualized. The rate of implantation in the pregnant group was 58.1% (18/31). CONCLUSION(S) These results provide evidence for the benefits of extending human embryo culture with P1 and blastocyst media for all normally fertilized embryos in vitro.

Effects of semen storage and separation techniques on sperm DNA fragmentation

OBJECTIVE To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. DESIGN Controlled clinical study. SETTING An assisted reproductive technology laboratory. PATIENT(S) Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. INTERVENTION(S) One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. MAIN OUTCOME MEASURE(S) DNA fragmentation as measured by SCD. RESULT(S) There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. CONCLUSION(S) The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use.

Should the Gynecologist Perform Laparoscopic Bowel Resection to Treat Endometriosis? Results Over 7 Years in 168 Patients

STUDY OBJECTIVE To assess the feasibility and safety of laparoscopic bowel resections for endometriosis performed by gynecologic surgeons. DESIGN Retrospective cohort study (Canadian Task Force Classification II-3). SETTING Fertility and pelvic surgery clinics. PATIENTS One hundred sixty-eight women (age 21-53 years) with symptoms including pelvic pain, infertility, or both with 252 bowel endometriotic lesions underwent laparoscopic bowel resection performed by gynecologic surgeons between May 2000 and January 2008. INTERVENTIONS Laparoscopic procedures for excision of several endometriotic nodes and lesions included shaving resection (LscShR), discoid resection (LscDR), segmental resection (LscSgR), terminal ileal resection (LscIR), partial cecal resection (LscCR), and appendectomy (LscAp). MEASUREMENTS AND MAIN RESULTS The 168 patients underwent 172 laparoscopic bowel resections (4 patients were operated on twice) by the same surgeon. Lesions were distributed as follows: 133 (79%) in the rectum, 61 (24%) in the sigmoid colon, 47 (19%) in the appendix, 5 (2%) in the terminal ileum, 3 (1%) in the descending colon, and 3 (1%) in the cecum. At surgeon discretion, 12 lesions were not resected. A total of 216 bowel procedures were necessary to remove the 240 lesions include shaving resection in 22 patients (10%), discoid resection in 52 (24%), segmental resection in 92 (42%), terminal ileal resection in 2 (1%), partial cecal resection in 1 (0.6), and appendectomy in 47 (22%). Major complications occurred in 13 patients (7.6%) and included rectovaginal fistula in 3 patients (1.7%), rectosigmoid anastomosis dehiscence and bowel occlusion in 1 patient each (0.6%), and persistent bowel dysfunction in 4 patients (2.3%). These results are comparable to those reported in the literature to date. Complete relief of symptoms (measured using the Visick scale) was noted in patients with dysmenorrhea (59%), dyspareunia (75%), noncyclic pelvic pain (90%), pain on defecation (100%), constipation (83%), and cyclic rectal bleeding (100%). CONCLUSION Surgery to treat bowel endometriosis can be safely and efficiently performed by the gynecologic pelvic surgeon. Meticulous training and a multidisciplinary approach to comprehensive operative care are necessary. These findings can be validated by prospective collaborative studies and reports from other surgeons.

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome.

BackgroundClaudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy.MethodsEndometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated.ResultsFour patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4-/LIF+); 20% showed strong CLDN4 and strong LIF (LIF+/CLDN4+); 28% showed strong CLDN4 and weak LIF (CLDN4+/LIF-); and 36% showed weak CLDN4 and weak LIF (CLDN4-/LIF-). Successful implantation after IVF was associated with CLDN4-/LIF+(p = 0.003). Patients showing this endometrial CLDN4-/LIF+ immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4+/LIF- immunolabeling (p = 0.007).ConclusionThe combined immunolabeling expression of CLDN4-/LIF+ in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

The benefits of finasteride for hirsute women with polycystic ovary syndrome or idiopathic hirsutism

Finasteride has been used frequently in the treatment of prostate hyperplasia ,but this drug inhibits 5α-reductase and for this reason could be useful for the treatment of hirsutism. The aim of this study was to evaluate the clinical and hormonal effects of finasteride on hirsute women with idiopathic hirsutism or polycystic ovary syndrome. Twenty-four women were randomly divided into two groups: those given placebo and those given finasteride 5 mg/day. The treatment period was 6 months. All patients were evaluated before the beginning of treatment (baseline) and after 3 and 6 months of treatment using clinical examination through Ferriman-Gallwey score ,blood pressure ,cardiac frequency and body mass index. Also ,we collected blood for hormonal determination of levels of prolactin ,17α-hydroxyprogesterone, follicle stimulating hormone ,luteinizing hormone ,total and free testosterone ,dehydroepiandrosterone sulfate ,androstenedione and dihydrotestosterone. Furthermore ,all patients were asked about their concerns and satisfaction with the treatment. The results showed that the Ferriman-Gallwey score in the 6th month of finasteride treatment was significantly lower than at baseline and the 3rd month of this drug treatment. The dihydrotestosterone level in the finasteride group was also significantly reduced compared to that in the placebo group. The other hormones did not show any statistical difference during the study. All the patients treated with finasteride perceived a reduction in hirsutism after 6 months. In conclusion ,our data suggest that finasteride may be effective for the treatment of the hirsute woman with idiopathic hirsutism or polycystic ovary syndrome.

Metoclopramide-Induced Hyperprolactinemia Affects Mouse Endometrial Morphology

Information about the effects of pituitary hyperprolactinemia on endometrium, especially in levels coexisting with absence of amenorrhea in women, is scarce. The interference of high prolactin levels on endometrial morphology was thus investigated in young post-pubertal and adult mice rendered hyperprolactinemic by long-term treatment with metoclopramide (MC). No remarkable differences have been noticed upon light microscopy examination of the endometria comparing young to adult cycling MC-treated mice, except on the max/min diameter ratio, which in young animals was lower than in adults (ANOVA, p < 0.01). Both young and adult MC-treated mice presented an increased number of endometrial glands than their respective controls (ANOVA, p < 0.01). However, young MC-treated animals showed the highest values of endometrial thickness index compared to other groups (ANOVA, p < 0.01). Our results indicate that MC-induced hyperprolactinemia causes mouse endometrium proliferation, mainly in young animals.

Effects of melatonin on histomorphology and on the expression of steroid receptors, VEGF, and PCNA in ovaries of pinealectomized female rats

OBJECTIVE To evaluate the effect of melatonin both on the ovaries of pinealectomized female rats through histomorphometric analysis and on steroid receptors, proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor (VEGF) expression. DESIGN Experimental study. SETTING Federal University of São Paulo, Brazil. ANIMAL(S) Forty female rats. INTERVENTION(S) Forty rats were divided equally into four groups: GI-vehicle without surgery; GII--surgery without removal of the pineal gland (sham); GIII--pinealectomized with vehicle; and GIV--pinealectomized with melatonin treatment. After treatment for 3 consecutive months, the animals were killed and their ovaries removed for analysis. MAIN OUTCOME MEASURE(S) Estrogen and progesterone receptors, histologic and immunohistochemical analysis. RESULT(S) The GIII samples presented signals of proliferation on ovarian surface epithelium and interstitial cells as well as high expressions of PCNA and VEGF in those structures compared with GI, GII, and GIV. Also, the levels of progesterone receptor (fmol/g) in ovaries of GIII (250.6 ± 32.4) were significantly lower than in those of GI (429.0 ± 23,8), GII (442.3 ± 30.2), and GIV (564.1 ± 78.7). The levels of progesterone in GIII were superior to those in GI, GII, and GIV. CONCLUSION(S) Our findings suggest that melatonin may attenuate proliferation in ovarian structures and increase the number of luteal bodies as well as the levels of progesterone receptor.