Best Practice Recommendations for Infertility Management.

Aim: The objective of this document is to provide guidance to the infertility specialist, gynecologist, embryologist, and counselors on the management of sub-fertility and brief them with the recent advances in the field. These recommendations will aid the aforementioned healthcare professionals in everyday clinical decisions about appropriate and effective care of their patients with the best available evidence. Participants: Extensive deliberations, discussion, and brainstorming was done between different reproductive medicine (RM) specialists, to develop the recommendations. Evidence: A systematic review of the literature published up to June 2019 was carried out using PubMed and Cochrane Collaboration Library. International guidelines, cohort studies, case series, observational studies, and randomized controlled trials currently available in the literature were reviewed. Indian data whatever available was also reviewed. Process: Primary meetings were held with leading reproductive medicine specialists. Each topic was brainstormed on by a group of reproductive medicine experts, who then prepared the first draft of the recommendation. These recommendations then were reviewed by Dr. Jaideep Malhotra, Dr. Gouri Devi, and Dr. Madhuri Patil along with the chief co-ordinator of each consensus to finalize the final draft. Conclusions: From the literature and discussion of the available evidence, several topics were identified for which evidence is inconsistent, insufficient, or non-existing. For the benefit of couples undergoing several treatments, the working committee recommends that future research, where possible in well-designed RCTs, will help in establishing evidence for a particular practice. In the Indian context, one also needs to take into consideration facilities and options available, cost, lack of insurance coverage, experimental nature of some advanced techniques used.

Direct impact of gonadotropins on glucose uptake and storage in preovulatory granulosa cells: Implications in the pathogenesis of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is often associated with higher levels of LH, and arrested ovarian follicular growth. The direct impact of high LH on FSH mediated metabolic responses in PCOS patients is not clearly understood. METHOD: In order to investigate the impact of FSH and LH on glucose metabolism in preovulatory granulosa cells (GCs), we used [U14C]-2 deoxyglucose, D-[U14C]-glucose or 2-NBD glucose to analyse glucose uptake and its incorporation into glycogen. To reproduce the high androgenic potential in PCOS patients, we administered hCG both in vitro and in vivo. The role of IRS-2/PI3K/Akt2 pathway was studied after knockdown with specific siRNA. Immunoprecipitation and specific assays were used for the assessment of IRS-2, glycogen synthase and protein phosphatase 1. Furthermore, we examined the in vivo effects of hCG on FSH mediated glycogen increase in normal and PCOS rat model. HEK293 cells co-expressing FSHR and LHR were used to demonstrate glucose uptake and BRET change by FSH and hCG. RESULTS: In normal human and rat granulosa cells, FSH is more potent than hCG in stimulating glucose uptake, however glycogen synthesis was significantly upregulated only by FSH through increase in activity of glycogen synthase via IRS-2/PI3K/Akt2 pathway. On the contrary, an impaired FSH-stimulated glucose uptake and glycogen synthesis in granulosa cells of PCOS-patients indicated a selective defect in FSHR activation. Further, in normal human granulosa cells, and in immature rat model, the impact of hCG on FSH responses was such that it inhibited the FSH-mediated glucose uptake as well as glycogen synthesis through inhibition of FSH-stimulated IRS-2 expression. These findings were further validated in HEK293 cells overexpressing Flag-LHR and HA-FSHR, where high hCG inhibited the FSH-stimulated glucose uptake. Notably, an increased BRET change was observed in HEK293 cells expressing FSHR-Rluc8 and LHR-Venus possibly suggesting increased heteromerization of LHR and FSHR in the presence of both hCG and FSH in comparison to FSH or hCG alone. CONCLUSION: Our findings confirm a selective attenuation of metabolic responses to FSH such as glucose uptake and glycogen synthesis by high activation level of LHR leading to the inhibition of IRS-2 pathway, resulting in depleted glycogen stores and follicular growth arrest in PCOS patients.

Successful fertilization and embryo development after spermatid injection: A hope for nonobstructive azoospermic patients.

Spermatids are the earliest male germ cells with haploid set of chromosomes. Spermatid injection was introduced in human assisted reproduction for the treatment of men with non-obstructive azoospermia. Spermatozoa can be recovered in half of patients with nonobstructive azoospermia. The use of spermatids for intracytoplasmic injection (ICSI) has been proposed for cases in which no spermatozoa can be retrieved. However, there are low pregnancy rates following ICSI using round spermatids from men with no elongated spermatids or spermatozoa in their testes. The in vitro culture of immature germ cells has been proposed as a means to improve this poor outcome. Oocyte activation rarely occurs when injected with a spermatid. Therefore, spermatid injection requires use of calcium ionophores for oocyte activation which is otherwise carried out by PLC zeta from mature sperms. This is the only option available for the nonobstructive azoospermic patients to have their own biological child.

FSH stimulates IRS-2 expression in human granulosa cells through cAMP/SP1, an inoperative FSH action in PCOS patients.

Follicle stimulating hormone (FSH) plays a central role in growth and differentiation of ovarian follicles. A plethora of information exists on molecular aspects of FSH responses but little is known about the mechanisms involved in its cross-talk with insulin/IGF-1 pathways implicated in the coordination of energy homeostasis in preovulatory granulosa cells (GCs). In this study, we hypothesized that FSH may regulate IRS-2 expression and thereby maintain the energy balance in GCs. We demonstrate here that FSH specifically increases IRS-2 expression in human and rat GCs. FSH-stimulated IRS-2 expression was inhibited by actinomycin D or cycloheximide. Furthermore, FSH decreases IRS-2 mRNA degradation indicating post-transcriptional stabilization. Herein, we demonstrate a role of cAMP pathway in the activation of IRS-2 expression by FSH. Scan and activity analysis of IRS-2 promoter demonstrated that FSH regulates IRS-2 expression through SP1 binding sites. FSH stimulates SP1 translocation into nucleus and its binding to IRS-2 promoter. These results are corroborated by the fact that siRNA mediated knockdown of IRS-2 decreased the FSH-stimulated PI3K activity, p-Akt levels, GLUT4 translocation and glucose uptake. However, FSH was not able to increase IRS-2 expression in GCs from PCOS women undergoing IVF. Interestingly, IRS-2 mRNA expression was downregulated in GCs from the PCOS rat model. Taken together, our findings establish that FSH induces IRS-2 expression and thereby activates PI3K, Akt and glucose uptake. Crucially, our data confirms a molecular defect in FSH action in PCOS GCs which may cause deceleration of metabolism and follicular growth leading to infertility. These results lend support for a therapeutic potential of IRS-2 in the management of PCOS.

Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis.

CONTEXT: Polycystic ovary syndrome (PCOS) is a heterogeneous, genetically complex, endocrine disorder of uncertain etiology in women. OBJECTIVE: Our aim was to compare the gene expression profiles in stimulated granulosa cells of PCOS women with and without insulin resistance vs. matched controls. RESEARCH DESIGN AND METHODS: This study included 12 normal ovulatory women (controls), 12 women with PCOS without evidence for insulin resistance (PCOS non-IR), and 16 women with insulin resistance (PCOS-IR) undergoing in vitro fertilization. Granulosa cell gene expression profiling was accomplished using Affymetrix Human Genome-U133 arrays. Differentially expressed genes were classified according to gene ontology using ingenuity pathway analysis tools. Microarray results for selected genes were confirmed by real-time quantitative PCR. RESULTS: A total of 211 genes were differentially expressed in PCOS non-IR and PCOS-IR granulosa cells (fold change≥1.5; P≤0.001) vs. matched controls. Diabetes mellitus and inflammation genes were significantly increased in PCOS-IR patients. Real-time quantitative PCR confirmed higher expression of NCF2 (2.13-fold), TCF7L2 (1.92-fold), and SERPINA1 (5.35-fold). Increased expression of inflammation genes ITGAX (3.68-fold) and TAB2 (1.86-fold) was confirmed in PCOS non-IR. Different cardiometabolic disease genes were differentially expressed in the two groups. Decreased expression of CAV1 (-3.58-fold) in PCOS non-IR and SPARC (-1.88-fold) in PCOS-IR was confirmed. Differential expression of genes involved in TGF-ß signaling (IGF2R, increased; and HAS2, decreased), and oxidative stress (TXNIP, increased) was confirmed in both groups. CONCLUSIONS: Microarray analysis demonstrated differential expression of genes linked to diabetes mellitus, inflammation, cardiovascular diseases, and infertility in the granulosa cells of PCOS women with and without insulin resistance. Because these dysregulated genes are also involved in oxidative stress, lipid metabolism, and insulin signaling, we hypothesize that these genes may be involved in follicular growth arrest and metabolic disorders associated with the different phenotypes of PCOS.