Failed ovarian cortex transplant but successful ovulation induction in a cancer survivor with premature ovarian failure: A paradox.

Better diagnostic and treatment modalities for malignancies occurring in childhood and young age have increased the overall survival of the affected young girls and boys, but this has come at a cost of developing premature ovarian failure in girls and azoospermia in boys because of the gonadotoxicity of chemotherapy and radiotherapy. Thus, young girls and women of reproductive age who are at risk of ovarian failure due to cancer treatment must be mandatorily offered fertility preservation in any form such as cryopreservation of mature metaphase II oocytes after ovarian stimulation with gonadotropins and oocyte recovery and cryopreservation of embryos if the young female has a partner. However, these modalities may require the postponement of chemotherapy. Thus, in such instances, cryopreservation of ovarian tissue remains the only option. Herein, we report a case of a nulligravid young female patient who had stage IV Hodgkin's lymphoma and had to be taken up for immediate fertility preservation owing to the advanced stage of the disease. Therefore, cryopreservation of ovarian tissue was performed. The uniqueness of the case is that after remission of the disease was achieved with chemotherapy, transplantation of the ovarian tissue was carried out which was not successful, but the desire for motherhood prompted to carry out an ovulation induction in the remnant native ovary with a severely compromised ovarian reserve, and she conceived.

ISAR Consensus Guidelines on Safety and Ethical Practices in In vitro Fertilization Clinics.

STUDY QUESTION: What are the Safe and Ethical practices for ART applicable in INDIA? WHAT IS ALREADY KNOWN: The Indian IVF industry is booming; with mushrooming of assisted reproductive technology (ART) clinics in the country, the need for regulation is immense. The ISAR has taken up this initiative to lead the way forward in establishing practice guidelines for the safe and ethical use of ARTs in our country. These guidelines discuss the points to consider before the starting of an IVF unit, to the designing of the laboratory, the staffing pattern and experience recommendations, laboratory safety guidelines, documentation and patient traceability, gamete traceability, handling biological material, the consumables and media, and different consents and checklists and also propose key performance indicators for the Indian scenario. STUDY DESIGN SIZE DURATION: This is the report of a 2-day consensus meeting where two moderators were assigned to a group of experts to collate information on safe and ethical ivf practices in INDIA. This meeting utilised surveys, available scientific evidence and personal laboratory experience into various presentations by experts on pre-decided specific topics. PARTICIPANTS/MATERIALS SETTING METHODS: Expert professionals from ISAR representing clinical and embryology fields. MAIN RESULTS AND THE ROLE OF CHANCE: The report is divided in various components including the regulations, the various requirements for an ART center, qualifications and trainings, recommendations on good practices and quality management: the report and recommendations of the expert panel reflect the discussion on each of the topics and try to lay down good practice points for labs to follow. LIMITATIONS REASONS FOR CAUTION: The recommendations are solely based on expert opinion. Future availability of data may warrant an update of the same. WIDER IMPLICATIONS OF THE FINDINGS: These guidelines can help labs across the country to standardise their ART services and improve clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: The consensus meeting and writing of the paper was supported by funds from CooperSurgical India.

Correlation between Serum Progesterone Level on the Day of Ovulation Trigger During In vitro Fertilization and Its Effect on Treatment Outcome.

BACKGROUND: Premature luteinization (PL) is defined as a premature rise in serum progesterone concentration on or before the day of ovulation trigger with human chorionic gonadotropin. The incidence of PL varies between 5% and 30% during in vitro fertilization and embryo transfer (IVF-ET). MATERIALS AND METHODS: The prospective observational study comprising 380 patients undergoing IVF-ET. Blood samples were collected for serum progesterone level estimation on the day of ovulation trigger. Ovum pickup was done 36 h later and serum progesterone levels were correlated with IVF-ET outcome. STUDY OUTCOME: To correlate serum progesterone level on the day of ovulation trigger during IVF and its effect on treatment outcome. RESULTS: Mean serum progesterone level in the positive pregnancy group and negative pregnancy group was 0.892 ± 0.752 ng/ml and 0.91 ± 0.688 ng/ml, respectively (P = 0.961). The overall incidence of PL was 12.8% with 12.7% and 13.6% in the agonist and antagonist protocol respectively (P = 0.9001). PL incidence was 13.5% and 13.4% in positive pregnancy and negative pregnancy group (P = 0.223). CONCLUSION: PL has been associated with 12.8% of the IVF cycles. There was no statistically significant difference observed in the incidence of PL between different IVF stimulation protocols. PL does not seem to affect IVF outcome.

Diagnostic Office Vaginohysteroscopy in Evaluation of Infertility Prior to IVF: A Retrospective Analysis of 1000 Cases.

AIM: The aim of this study was to analyze the utility of routine use of diagnostic office vaginohysteroscopy in the evaluation of uterine cavity in infertility patients prior to IVF-ET. MATERIALS AND METHODS: We conducted a retrospective analysis of 1000 women who had undergone routine diagnostic office vaginohysteroscopy as an institutional protocol in the evaluation of infertility prior to IVF-ET cycle at a tertiary care hospital. They were divided into two groups: primary infertility (group I) and secondary infertility (group II). The primary outcome was the finding of an abnormal uterine cavity (congenital abnormality vs acquired abnormality). RESULTS: One thousand women underwent routine diagnostic office vaginohysteroscopy in the evaluation of infertility prior to IVF-ET. There were no intraoperative or postoperative complications. Vaginohysteroscopy revealed an abnormal uterine cavity in 13.8% (1000 patients) of women. Primary infertility group (I) had 13.19% (811 patients), and secondary infertility group (II) had 16.4% (189 patients) abnormal uterine cavities. CONCLUSION: Diagnostic office vaginohysteroscopy has a definite role in the uterine cavity evaluation in infertility patients prior to IVF, but routine use should not be recommended considering the low incidence of abnormal uterine cavity findings. Moreover, the majority of these uterine cavity abnormalities can be detected by less invasive tests such as HSG, TVS, SSG and 3D ultrasound.

Chapter 7 Human Epididymal and Testicular Sperm Cryopreservation.

Since the advent of intracytoplasmic sperm injection (ICSI) in the early 1990s, surgical techniques to recover samples from the epididymis and testis directly have been used to benefit patients suffering from obstructive and nonobstructive azoospermia. Various studies have demonstrated comparable fertilization, ongoing pregnancy, and implantation rates when fresh and frozen-thawed epididymal sperms were used for ICSI [1]. Injection of fresh and frozen testicular sperms into mature oocytes resulted in similar fertilization rates in cases of obstructive azoospermia. However, in cases of nonobstructive azoospermia, the outcome depends upon the degree of impairment of spermatogenesis, criteria for sperm freezing, and patient selection [2].

Sperm function test.

With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization.

Study of human sperm motility post cryopreservation.

BACKGROUND: Cryopreservation of spermatozoa is a widely used technique to preserve the fertility of males. It can also benefit the armed forces personnel who are to be sent for long recruitments, while leaving their families behind. This study, apart from studying the effects of freezing and thawing, reveals the effect of the post thaw interval on the motility of the human spermatozoa and thus widens the insemination window period. METHODS: A detailed semen analysis was carried out as per the WHO guidelines for 25 samples. The samples were then washed, analysed and frozen in liquid nitrogen. The semen samples were subsequently thawed and similarly analysed after 20 min and 40 min of thawing. This was then followed by statistical analysis of the comparative motilities. RESULTS: Motility of sperms is found to decrease after cryopreservation. However, the study revealed that after thawing a significant increase in the motility of the sperms was noted with the progression of time (p < 0.05). CONCLUSION: By simulating conditions similar to the in vivo conditions for the post thaw semen samples, we can safely wait, confirm the parameters like motility and count, and then inseminate the samples instead of blindly inseminating them immediately after thawing.

VEGF antagonist for the prevention of ovarian hyperstimulation syndrome: Current status.

Ovarian Hyperstimulation Syndrome (OHSS) an iatrogenic and potentially life-threatening complication resulting from an exaggerated response to ovulation induction with gonadotropins during assisted reproductive technologies, is a self-limiting disorder with a broad spectrum of clinical manifestations related to increased capillary permeability and fluid retention brought about by many biochemical mediators especially vascular endothelium growth factor (VEGF), playing a pivotal role in its pathophysiology. Although various strategies had been proposed and tried to prevent this serious complication none was found to be completely effective. With the current knowledge and understanding of the causative molecule i.e. VEGF in the pathogenesis of OHSS, pharmacologic tools targeting this member of the family of heparin binding proteins, seems promising. Antagonizing and blocking VEGF action by dopamine agonists especially Cabergoline has shown to be a valid alternative to overcome the changes induced by the gonadotropins. Delaying embryo transfer with embryo cryopreservation definitely reduces the incidence of OHSS but not the early OHSS. In-vitro maturation of oocytes a major breakthrough in the field of ART although totally eliminates the risk of OHSS is highly labor intensive and cannot routinely carried in all cycles. Thus the newer drugs, mainly the dopamine agonists in the light of the new pathogenic and pharmacological evidence, should definitely be considered for prevention of both early and late OHSS.

Mitochondrial DNA variations in ova and blastocyst: implications in assisted reproduction.

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.

Systemic methotrexate: An effective alternative to surgery for management of unruptured ectopic pregnancy.

BACKGROUND: Medical management of an unruptured ectopic pregnancy with intramuscular methotrexate is a common and cost-effective alternative to surgery. Early diagnosis and timely institution of methotrexate has resulted in a dramatic decline in the morbidity, mortality and financial burden associated with ectopic pregnancy. The objective of our study was to evaluate the efficacy of medical management in a series of patients with unruptured ectopic pregnancy meeting the strict inclusion criteria. METHODS: Multiple dose methotrexate regimen was utilized for the management of the cases. Parenteral methotrexate 1 mg/kg was administered on Day 1, Day 3, Day 5 and Day 7 whereas rescue dose of injection leucovorin 0.1 mg/kg was given on Day 2, Day 4, Day 6 and Day 8. Monitoring for the resolution of the ectopic was carried out with ß-hCG, done on Day 1, Day 3, Day 5, and Day 7. Any value showing a decline of >15% of the baseline value led to the termination of treatment and only surveillance was carried out. If the decrease was <15%, treatment was continued. RESULTS: Total 43 patients with ectopic pregnancy were diagnosed over a period of 1 year, conceived during infertility evaluation and treatment. 11 cases went directly for surgery whereas 32 were subjected to medical treatment. 75% of the patients got successfully treated and rest 25% required surgery following failed medical management. CONCLUSIONS: Methotrexate therapy is a safe and effective alternative for the management of unruptured ectopic pregnancies with minimal or no side-effects and associated advantage of avoiding invasive surgery.

Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies.

PROBLEM: To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. METHOD OF STUDY: Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. RESULTS: Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P < 0.05) inhibition in the baculovirus-expressed recombinant ZP4-mediated acrosome reaction. MA-1671 recognized N-terminal fragment of ZP4 and minimal epitope mapped to amino acid residues 126-130 (PARDR), whereas MA-1662 reacted to C-terminal fragment and minimal epitope mapped to amino acid residues 256-260 (ENELV). CONCLUSIONS: The epitopes corresponding to both N- and C-terminal parts of human ZP4 may be relevant for its biological activity.

Embryo reduction: our experience.

BACKGROUND: The transvaginal ultrasound-guided embryo reduction technique is a feasible option for the prevention and management of the medical and obstetric risks associated with high-order multiple pregnancy resulting from assisted reproductive treatment. METHOD: Multifoetal pregnancy reduction was carried out in 51 in vitro fertilisation pregnancies (IVF) and one intrauterine quintuplet pregnancy resulting from intrauterine insemination (IUI) using transvaginal approach under ultrasonographic guidance. RESULTS: Of the 52 embryo reduction procedures, 48 (92%) were performed between the seventh and eighth weeks of gestation, three between eighth and ninth weeks and one in the 10th week of gestation. Forty-nine patients (94%) underwent reduction from triplets to twins, two from quadruplet to twins, and one from quintuplet to twin pregnancy. The average time required for the embryo reduction was 5.0 ± 0.5 minutes per sac in early gestation (6th-9th weeks), increasing to 8.5 minutes per sac for later procedures, due to technical difficulties brought about by increased embryo size and mobility. All embryo reduction procedures were successfully performed in a single session. CONCLUSION: Transvaginal ultrasound guided embryo reduction technique performed between seventh and eighth-weeks of gestation is an effective and safe procedure for embryo reduction.

Does catheter choice during embryo transfer alter the pregnancy rate?

BACKGROUND: Although every step in the in vitro fertilisation (IVF) procedure is important, the impact of embryo transfer (ET) on pregnancy rate (PR) is significant. Of all the crucial aspects of ET the type of catheter used and the technique of transfer on the PR has drawn the maximum attention and controversy. We aimed to compare the outcome of two different ET catheters on the PR. METHOD: A prospective analysis comparing the classical Frydman (Laboratoire CCD, France) and the soft Cook (Cook Medical, Indiana, USA) ET catheters was performed. Primary end-point was clinical pregnancy rate (CPR); secondary end-points were rates of difficult transfer. A total of 1,446 ETs were performed in women undergoing IVF treatment, of which 723 cycles were randomised to the Cook catheter and 723 to the Frydman catheter. RESULTS: It was observed that, although the Cook catheter was related to a slightly higher PR, the overall comparison failed to indicate a significant difference in CPR. It was also seen that the ease of transfer did not significantly affect the PRs. CONCLUSION: Individual variables during ET may not contribute significantly to the success of an IVF programme; however, a holistic approach encompassing all the factors is quintessential to improve the PR.

Nucleotide variations in mitochondrial DNA and supra-physiological ROS levels in cytogenetically normal cases of premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is defined as the cessation of ovarian function under the age of 40 years and is characterized by amenorrhea, hypoestrogenism, and elevated serum gonadotrophin concentration (FSH). It is a heterogeneous disorder with a multicausal pathogenesis; however, majority of cases are idiopathic. In idiopathic POI, involvement of unknown mechanisms may increase rate of oocyte apoptosis. Studies have shown that elevated reactive oxygen species (ROS) levels affect the quality of gametes. Mitochondrial mutations in different complexes of electron transport chain have been reported to disrupt the electron flow which lead to formation of more superoxide ions or increased levels of ROS. This study was aimed to screen the mitochondrial genome for variations in idiopathic POI (n = 25) and occult ovarian insufficiency (OI) (n = 5) patients. 30 patients diagnosed with POI and occult OI were enrolled in this study. Blood samples were collected from the patients and controls. DNA was extracted using phenol chloroform method. A total of 102 nucleotide variations were observed in patients as compared with 58 nucleotide variations in controls. 24% variations were found to be non-synonymous and 76% were synonymous. It was found that 48% variations were in complex I, 8% in complex III, 24% in complex IV, and 20% in complex V of electron transport chain. We found most of the non-synonymous mitochondrial variations in complex I (48%) of the respiratory chain which is the largest enzyme complex and is associated with oxidative stress. Some non-synonymous pathogenic alterations (p.M31T, p.W239C, p.L128Q) and non pathogenic alterations (ATPase6:p.T53I, ATPase6:p.L190F, ATPase6:p.L199L) were found to be significantly higher in cases as compared with controls. The preliminary data suggest that the mitochondrial mutations and subsequent decline in ATP levels may accelerate follicular atresia and lead to POI. The results of this preliminary study highlight the need to extend this study by analyzing large number of samples in different ethnic populations and analyze for ROS levels and mitochondrial mutations in oocytes as they are of different embryonic origin and develop in a different microenvironment.

Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida.

BACKGROUND: Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction. METHODS: Heat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP. RESULTS: SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction. CONCLUSIONS: Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.