Anahita R. Chauhan

Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture

BackgroundCryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells.MethodsSerum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR.ResultsA prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition.ConclusionThe present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

BackgroundRecent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro.MethodsOvarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging.ResultsDifferential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming.ConclusionsPresence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine.

Panel of Autoimmune Markers for Noninvasive Diagnosis of Minimal–Mild Endometriosis A Multicenter Study

Endometriosis, characterized by the presence of endometrial-like tissue at extrauterine sites, is a common, chronic, estrogen-dependent, inflammatory condition associated with pelvic pain, subfertility, dysmenorrhea, and dyspareunia, affecting about 10% of reproductive-age women in any population. The diagnosis of endometriosis is usually delayed on an average by 8 to 11 years leading to significant consequences in terms of disease progression. The current study was aimed to validate enzyme-linked immunosorbent assay based on the epitopes of stomatin-like protein 2, tropomodulin 3 (TMOD3), and tropomyosin 3 (TPM3) for diagnosis of minimal–mild endometriosis (revised American Fertility Society Classification (rAFS) stage I-II) and to compare the performance with the reported markers: cancer antigen (CA) 125, CA19-9, α-enolase, Serine/threonine-protein kinase (PDIK1L), and syntaxin 5. This was a cross-sectional, multicenter study conducted during the year 2012 to 2015. Women with minimal–mild endometriosis (rAFS stage I-II [n = 133]) and healthy controls (n = 104) were screened for 11 novel autoimmune markers and reported markers α-enolase, PDIK1L, syntaxin 5, CA-125, and CA19-9. The sensitivity and diagnostic accuracy of serum antibodies against all the 11 epitopes were higher than that of CA-125, CA19-9, α-enolase, PDIK1L, and syntaxin 5 for diagnosis of rAFS stage I to II endometriosis. The sensitivity of 6 biomarkers (anti-TMOD3b-autoAb, anti-TMOD3c-autoAb, anti-TMOD3d-autoAb, anti-TPM3a-autoAb, anti-TPM3c-autoAb, and anti-TPM3d-autoAb) was higher at the specificity of ≥80% for diagnosis of rAFS stage I to II endometriosis as well as ultrasound-negative endometriosis. Further, logistic regression models of this panel of biomarkers showed increase in sensitivity, specificity, and diagnostic accuracy than individual biomarkers. The panel of 6 autoimmune biomarkers could be useful in setting up of noninvasive diagnostic test for detection of minimal–mild endometriosis.

Novel FSH receptor mutation in a case of spontaneous ovarian hyperstimulation syndrome with successful pregnancy outcome

The objective is to study the FSH receptor (FSHR) for mutations in a case of spontaneous ovarian hyperstimulation syndrome (sOHSS). This is a single case study and it examined patient who presented with spontaneous critical OHSS in early pregnancy and had successful good obstetric outcome. Intervention of this study was analysis of blood for genetic analysis of FSHR postdelivery. The main outcome measure noted was FSHR mutation. The study resulted in a novel, here though unreported, heterozygous mutation in FSHR gene at nucleotide position 1346 (AC 1346 T to AAT) in exon 10 yielding a threonine to asparagine (Thr 449 Asn) substitution in the transmembrane domain helix 3 of the FSHR. To conclude FSHR gene analysis can add to our understanding of sOHSS.

The “Saturday Effect” in Obstetrics: A Comparison Between Referral Patterns on Saturday and Other Days of the Week

Through this communication, we wish to highlight that a disproportionate number of obstetric patients are referred from the firstor second-level facilities to tertiary centres on weekends, especially Saturdays [1]. Day-of-the-week linked referral patterns are recognised in Western literature, not necessarily only in obstetrics [2]. However, documentation in contemporary Indian literature is not extensive. Our research question aimed to find if ‘‘taking a break during weekends’’ by doctors leads to increased inter-institutional referrals. We conducted a prospective observational study at our tertiary-care hospital over 1 year to ascertain if differential referral patterns exist. All obstetric cases referred on Saturdays were compared to those referred on all other days, including Sundays; data regarding reasons for referral, consistency between indication for referral and actual diagnosis after admission; and maternal and perinatal mortality patterns were analysed. Of the total 8271 confinements, referrals constituted 38.12%; however on Saturdays, 46.49% cases were referred as compared to only 25.36% on other days, which was statistically significant. There was also statistically significant difference between reasons for referral on Saturdays versus other days, with referral for non-medical reasons predominating on Saturdays (non-availability of doctors and NICU). Noteworthy is that almost half the cases managed on Saturdays were referred. (Table 1). In our country, most doctors in public sector work on Saturdays, at least in the morning. To look for differences in referrals based on time of day, 24-h time period from 7 am was divided into 3-h intervals and the time of arrival of referred cases was noted. Majority of patients were referred between 4 pm on Saturdays and 1 am on Sundays, as compared to a more uniform distribution among all time periods on other days; this was statistically significant (p value = 0.0347). Major inconsistencies were observed between reason for referral and actual diagnosis, mainly need for NICU and PIH. 25 and 19.5% of cases referred on other days of the week for presumed need for NICU or a diagnosis of PIH, respectively, did not need NICU nor had PIH; this number increased to 35 and 30% on Saturdays, respectively, for NICU and PIH. This highlights unnecessary transfers on Saturdays. LSCS rate was similar, 32.2 and 28% on Saturdays and other days. Perinatal and maternal mortality rates were comparable other days. The commonest causes Anahita R. Chauhan is a Professor and HOU in Department of Obstetrics and Gynecology at Seth GS Medical College and KEM Hospital, Mumbai. Sumit Chamariya is a Registrar in Department of Obstetrics and Gynecology at Seth GS Medical College and KEM Hospital, Mumbai. Madhva Prasad is a Assistant Professor in Department of Obstetrics and Gynecology at Seth GS Medical College and KEM Hospital, Mumbai.

Cesarean Myomectomy: Necessity or Opportunity?

Fibroids in pregnancy are increasingly common, due to advanced maternal age, better diagnostic tools and use of assisted reproductive techniques. Cesarean section (CS) is the commonest mode of delivery in these patients. Cesarean myomectomy (CM) is the term used to describe the removal of fibroids at CS; it has always been a controversial topic, with two schools of thought. Some obstetricians advise against it due to the traditional fear of massive obstetric hemorrhage and its attendant complications. However, recent literature advocates elective or opportunistic myomectomy in well-selected cases during CS. This is especially valuable in low-resource settings where the patient may be spared a repeat surgery and problems of anesthesia and cost associated with it. This review examines the recent published data on CM, its indications, technique, safety and applicability in modern obstetrics.

Virilization Secondary to Androgen-Secreting Tumor of the Ovary: A Report of Three Cases and Review of Literature

Androgen-secreting tumors of the ovary represent\ 5% of all ovarian tumors and are a rare cause (0.2% of cases) of androgen excess in females. They belong to the category of sex-cord stromal tumors; the most commonly implicated are Sertoli–Leydig cell tumors (SLCTs), Leydig cell tumors, steroid cell tumors, fibrothecomas and rarely granulosa cell tumors [1, 2]. Owing to their rarity, there is limited understanding of their natural progression and optimal management strategies. This report describes three cases and sheds light on diagnosis and treatment of these rare tumors.

Pregnancy in breast cancer survivor with anthracycline induced cardiomyopathy

With earlier diagnosis and improved treatment modalities and management of breast cancer patients, survival is improving. An increasing number of survivors are in the reproductive age group; however a neglected medical area is contraceptive advice, failure of which can result in unwanted pregnancy and further medical complications. An undiagnosed pregnancy in a breast cancer survivor with known anthracycline- induced cardiomyopathy is presented here.