Showing posts with label Pregnancy. Show all posts
Showing posts with label Pregnancy. Show all posts

Wednesday, August 7, 2024

Analysis of IVF Technology: Hope for Infertile Couples -Juniper Publishers

 

Gynecology and Womens Health - Juniper Publishers

Abstract

This research paper is designed to gain knowledge about the ART (Assisted Reproductive Technology) technique i.e IVF. Most of the population has a high prevalence of infertility but due to lack of awareness they fail to seek medical treatment, thus the main objective of this research is to help people understand IVF and clear their concepts. Worldwide 10% of all couple’s face infertility which is causing stress and further lead to depression and social isolation as it is the natural human desire to have offspring. Infertility is caused by many reasons explaining why fertility treatments are necessary. Infertility is defined as being unable to get pregnant even when no contraceptive measures taken. From a literature point of view, infertility is defined as a complex health issue that is not only due to blocked fallopian tubes, ovaries, or any other issue but can be also due to certain lifestyles i.e., stress, an age that leads towards the assisted reproductive technology. Science has played a major role in the development of assisted reproductive technology; ART is a reproductive technology that is curing both male and female infertility. Almost 40 years ago world’s first IVF baby was born, more than over 8 million babies are born through infertility treatment IVF as it is an infertility procedure in which egg and sperm are fertilized outside the body. This research is based on qualitative analysis and content analysis and library research method is used. According to an estimated report, 400 million individuals would be the result of IVF and other fertility treatments. IVF provides hope to millions of infertile couples with the pleasure of parenthood. Further advancements and development are on the way for IVF to make it more effective and improve its success rate.

Keywords: Assisted Reproductive Technology; In Vitro Fertilization; Fertility treatment; Infertile; Pregnancy

Introduction

Miracle Technology – IVF

It is an established fact that women are the much-deprived part of society and this attitude is reflected throughout our society. Similarly, it is seen that women are considered less important than men in all societies globally and the most ignored sectors are education, employment, health, etc. Among all the social aspects in which women face inequality, health is the major concern for women, which should be seriously addressed [1-5].

In 1994 Cairo organized the 4th International Conference on Population and Development which demanded universal access to reproductive health, but it was neglected by the Millennium Development Goals. Women are usually neglected in the health sector; due to this inequity, they face a great challenge in basic health facilities. Reproduction is an essential human desire, when a couple fails to conceive naturally it causes a great deal of psychological pressure by the society and culture towards infertile couple which is usually devasting and highly affects their well- being, as infertility and its treatment have a stressful effect on the couple. Medical techniques have changed dramatically during the last few decades, yet the transformation with the help of humans will be restricted. Arthur C. Clarke, the visionary and science fiction writer, famously said that any remarkable advancement in technology is unlike magic [6-10].

Whenever technology will get a boost and there will always be solutions to the desired problems. Medical techniques for infertility have brought a drastic change in the life of an infertile couple. In vitro fertilization or IVF is a globally accepted procedure helping infertile couples to have their own children. In the field of science, the arrival of In Vitro fertilization procedure was a milestone for the infertile couple as the infertility rate in the world is high. IVF is a miracle procedure for many couples dreaming to conceive a healthy child of their own and fulfilling their dream of becoming parents hence, IVF brings reality to a dream of parenthood that was not possible as it enhances the chances of conceiving [11-15].

According to WHO globally about 48 million couples and 186 million individuals face infertility, a reproductive collapse to attain pregnancy after regular unprotected sexual intercourse. Many couples face infertility with no symptoms unless they start trying to conceive, since infertility is not a disease and can be treated in today’s era of science. Several conditions can cause infertility in both men and women, the signs and symptoms of each can vary. Couples may face trouble in conceiving, but the medical industry has been forever making advances in this field. About 60 million people around the developing world face infertility. In 2004 about 186 million women in developing countries were facing primary and secondary infertility, WHO-DHS Comparative Report reveals. The situation in Pakistan is not different as primary infertility is 3.9%, while secondary infertility is 18.0% [16-20].

Assisted reproductive technology (ART) is a fertility treatment that can bring hope to couples with difficulties or an inability to conceive children. Techniques of ART increase the chance of successful pregnancy by the manipulation of eggs, sperm, or embryos. It is a technique for infertile couples who have already tried other infertility treatments but failed to conceive. Different risks associated with ART procedure include multifetal gestations, prematurity, low birth weight, small for gestational age, perinatal mortality, caesarean delivery, placenta previa, abruptio placentae, preeclampsia, and other birth defects [21-25].

The infertility rate is increasing in the world, but the arrival of In Vitro fertilization treatment was a breakthrough in the field of science and assisted reproductive technology as it turns out to be a miracle for struggling couples making their dream of parenthood into reality with the record of safe and successful deliveries. Factors may be affecting the effectiveness of IVF i.e specifically the age of the female partner’s medical history to consider. Couples conceived pregnancy through IVF faced anxiety and depression more than normal pregnancy conceived parents while after an unsuccessful IVF sadness, depression, and anger [26-30].

Cost of IVF

The charges for the basic cycle of Vitro fertilization (IVF) cycle is more than $12,000 but it varies from $15,000 - $10,000 which doesn’t include the cost of medications. Talking about the cost of IVF around the world varies as Greece has as low as 3000- 3500 EUR while the Czech Republic has 30 clinics managed by the Czech Society for Assisted Reproduction. Spain has the expensive treatment while Turkey is considered an alternative to European countries as it is cheaper than other countries. In Pakistan, it is out of reach for middle and lower-middle-class ranging from Rs.200000 -Rs.450000 (Figure 1).

Islamic Perspective

In Islam it is permissible to get infertility treatment with all assisted reproductive technologies, but the source of sperm, ovum and uterus must be from a legally married couple. Surrogacy is not permissible in Islam in fact frozen sperm is not allowed to use after the death of a husband. Quran and Hadiths have emphasized on the institution of marriage and family. Allah says in Quran in surah 16: 72; “And Allah has given you wives of your own kind, and has given you, from your wives, sons and grandsons, and has made provisions of good things for you. Is it then in vanity that they believe and in the grace of Allah that they disbelieve?”

Islam acknowledges that infertility is a significant hardship. The Quran gives the example of two prophets, Abraham and Zacharia, who were barren and described how they longed to have children of their own, even as they grew old and almost despaired of having children. Zacharia pray to his Lord, saying;

“0 my Lord! Grant unto me from You a progeny that is pure, for You are He that hears the Prayers.” The method they used to achieve their goal was to ask God repeatedly and sincerely with humility and faith. Eventually, God answered their prayers. “And the angels gave Abraham glad tidings of a son endowed with knowledge!”

Dr. Zaryab Setna a renowned gynaecologist in Pakistan states that what they practice in their clinic is according to Islam, no third person is involved, and eggs and sperms used for fertilization are from married couples [31,32].

Feminist Perspective

According to feminist researchers’ women are the primary source of IVF treatment as women receive more severe medical treatments which are riskier and prolonged as compared to their partners. According to Franklin, couples seek IVF treatment not only due to biological dysfunction but also to repair a missing societal role i.e parenthood [33].

Simone Bateman defines infertility as a pressure of society on couples to have their own family. It is estimated that the prevalence of infertility varies in the world about 1.5% of the female have primary fertility issues. According to feminist social pressures plays a crucial role in women’s life to fulfil their reproductive desires as these treatments are often psychologically and physically exhausting but they continue painful treatment to have their own child due to societal pressure. IVF is a source of hope to overcome the pain of infertility, as it is physically, emotionally, and financially draining for the couple.

Pakistani Perspective

In Pakistan, the first IVF centre was established in the mid of 1980s since then number of centres has been increased. In Pakistani society having a child is regarded as the only aim of being a woman, they are considered incomplete without a child. Infertility treatments are common in the world, but in Pakistani society, it is taboo to have any fertility treatment. IVF is still not considered a halal treatment in society as people don’t have knowledge about this treatment their concepts are not clear which makes it difficult for the couples seeking fertility treatment. Most of the couples who take IVF treatment do not want to disclose it to society as they are aware of the thoughts of society and they know society will judge them.

According to The Fertility clinic by Setna Dr. Zaryab Setna quoted that the success of IVF varies according to age and other factors i.e there are 41-42 % chances for age less than 35 years, 35% chances for women between 35-37 years, whereas 23-26% between age 38-40 years. According to a report on hospitals in Karachi i.e., the lack of knowledge about infertility explains the societal pressure on infertile couples.

The Agha Khan University report reveals that most of the time the couples face a lack of support from their families and a negative attitude from society led them to think even about suicide. The research concluded that infertility does not only socially isolate them, but the couples also face marital conflict as society pressurizes the newlywed to have immediate pregnancy as it is a common practice in a joint family system. According to another research, a woman is blamed for infertility in Pakistani society despite male infertility rates being higher. The research further stated that IVF is valid in Pakistan but is still not accepted as the permanent solution to fertility by society [34].

Review of Literature

Worldwide it is estimated that more than 8 million babies are born through in vitro fertilization (IVF), after the birth of the world’s first IVF baby 40 years ago. The discovery for the treatment of infertility by Robert G. Edwards is considered as the “greatest benefit to mankind”. The discovery of IVF has brought a drastic change in the field of reproductive medicine. About 2-3% of newborns are born through this technology in many countries. Robert G. Edwards was awarded Noble Prize for development in IVF, as the technique has been a source of joy for millions of infertile couples by giving them chance to conceive their own child.

In 2015 Ernst & Young’s report titled Expanding IVF treatment in India stated that nearly 10-15% of married couples are affected by infertility in India. The report further mentions that by the year 2020, couples aged between 30-40 years, are likely to be more affected by infertility.

Many couples seek help through fertility treatments to cope the infertility issues. In 70’s when initially IVF started the success rate was 5-10%, but with the passage of time and advancement of science and technology the success rate has increased now and the success rate per IVF cycle is 60-80%., though success is still not guaranteed, it is a ray of hope for those who have not conceived in years. According to Dr. Shweta Goswami, there is no guarantee of successful IVF for any infertile couple, because the success rate varies from patient to patient, it would be 10% for others or it would be 60%, but still, there is hope for them.

A 34-year-old female cancer patient gave birth to a baby by using a fertility procedure involving the collection, maturing, and freezing of eggs 5 years ago. It was a successful pregnancy by immature eggs that were later frozen and then matured. About 1-3% million births worldwide are the result of IVF in the U.S and Europe. The increased demand for IVF technology has led to research and development of the technology. According to a report (2010), an estimated 48.5 million couples were infertile i.e., 6 million increases since 1990. In 2002 according to Rustein and Shah, more than 186 million married women from developed countries (excluding China) were infertile. According to Dr. Zafar IVF is a treatment for infertility where other assisted reproductive technology fails to cure infertility. It is a hormone controlling ovulation process followed by removing eggs and sperm and then initiating the fertilization process in a fluid medium, later then the fertilized egg is transferred to the mother’s uterus for fetal fullterm development.

Research Methodology

Keeping in mind the research topic, the qualitative approach has been used as a research methodology. A method of content analysis was employed as it is a widely used qualitative research technique. The main purpose of this research was to have librarybased research including books and different articles and research from various libraries in Karachi and the main library and seminar libraries of the University of Karachi.

Conclusion

According to World Health Organization (WHO), infertility is stated as a disease of the reproductive system, that fails to achieve pregnancy after regular sexual intercourse. Infertility is a world problem that is growing continuously. According to Lewine, his analysis on extensive meta-regression draws attention to a significant decrease in sperm counts of fertile men from Europe, North America, Australia, and New Zealand.

Infertility knowledge is limited by lots of misconceptions and myths by society. A global survey was conducted, which revealed that many women have no knowledge about fertility treatment, most of the time regardless of seeking fertility treatment couples chose alternative medicines. Infertility treatment is usually a long and exhausting process for a couple and does not always have a happy ending for the couple but is still a ray of hope for the couple. Infertility in Pakistan is approximately 22% with 4% primary and secondary infertility is almost around 18%. Infertility should not be considered only a medical problem as it is also a social issue. Society must accept infertility treatments as a solution so that it can help couples to seek fertility treatment timely.

Medical science has made a remarkable success in the field of ART: IVF which has helped infertile couples to cope with fertility issues and help them to conceive their own child with the treatment. Furthermore, the modification of treatments will probably occur with ongoing use and practice. A typical IVF cycle lasts several weeks and involves many rounds of medication and minor surgical procedures in order to stimulate ovulation, pick up and fertilize gametes, and transfer embryos. Multiple pregnancies are the known complications of IVF as a result of the transfer of multiple embryos. Every couple wants to have a child of their own, ART is not common as people have no clear knowledge about IVF and its cultural and religious perspective is also not clear to the majority which makes it difficult for infertile couples to discuss and have awareness to help themselves with infertility. IVF is still out of reach for many couples as it is a very expensive treatment. Thus, like any other discovery or technology, IVF is a set of challenges and difficulties, but its result and success rate compensate for the challenges as it gives the pleasure of parenthood to millions of people.



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Thursday, November 9, 2023

A Case of Successful Cardiopulmonary Resuscitation in a Pregnant Woman with Eisenmenger’s Syndrome - Juniper Publishers

 Anesthesia & Intensive Care Medicine - Juniper Publishers

Abstract

Patients with Eisenmenger’s syndrome (ES) are advised to avoid pregnancy due to high maternal mortality, reaching 30 - 50%. A 37-year-old pregnant patient, at a gestational age of 14/15 weeks, was admitted to the Medical Center for abortion due to ES. Due to the complete presentation of the chorion, it was planned to perform curettage of the uterine cavity. After introduction of antibacterial drug in the perioperative period, acute decompensation of heart failure was observed, which required cardiopulmonary resuscitation and ECMO. This condition was assessed as an anaphylactic shock. It was possible to stabilize the patient after switching to ECMO. On the next day the patient was extubated and the ECMO system was explained on the 5th day. On the 29-th day the patient was discharged.

Keywords: Eisenmenger’s syndrome; Anaphylactic Shock; ECMO; Cardiopulmonary Resuscitation; Pregnancy

Introduction

Unfortunately, there are still several adult patients who did not manage proper cardiac surgical care in childhood and subsequent development of severe hemodynamic disorders later in their lives, up to the formation of Eisenmenger's syndrome (ES). ES is marked by the development of a shunt of blood from the right side to the left side of the heart, resulting in defects of systemic and pulmonary circulation, which occur due to the formation of severe pulmonary arterial hypertension (PAH). Patients with ES are advised to avoid pregnancy due to high maternal mortality [1], reaching 30 - 50% [2]. In accordance with current international clinical guidelines, pregnant women with ES should be treated in the following way. During pregnancy women with ES should be observed by a multidisciplinary team, in reference centers; specific therapy for PAH should be evaluated and possibly modified. Operative abdominal delivery is recommended. In the respective literature, several clinical cases of cardiopulmonary resuscitation (CPR) in patients with ES [3-6] are described. However, only one publication demonstrates a successful case of CPR [7]. The reason for the low effectiveness of CPR is associated with the pathophysiological features of ES. During heart massage, venous blood from the right chamber enters the left chamber of the heart, following into the systemic circulation.

Case Presentation

Patient M, 37 years old, pregnant, at a gestational age of 14/15 weeks, was admitted to the Almazov centre on June 27th of, 2022 for abortion due to ES. Informed consent was obtained from the people who participated in this clinical case.

Preoperative Management

From the anamnesis: patient is aware of her heart disease - ventricular septal defect (VSD) - since the age of 14. Patient has been repeatedly examined in cardiac surgery clinics, with surgical treatment denied due to high PAH. PAH-specific therapy was initiated (Bosentan was taken irregularly, and patient didn't switch to Sildenafil therapy).

Allergies: All antibacterial drugs that she received prior to her current hospitalization were well tolerated, without any anaphylactoid reactions. The patient’s weight was 52 kg, height was 149 cm. Blood pressure (BP) was 120/70 mm Hg. SpO2 was 78 - 81% at rest on atmospheric air.

The Main TTE Parameters: Left Atrium -35 mm; End-Diastolic Volume of the Left Ventricle (LV) - 51 ml; End-Systolic Volume of the LV- 21 ml; Stroke Volume - 30 ml; Ejection Fraction of the LV - 59%. Right Ventricular (RV) (4-chamber position) - 32 mm; RV Front Wall- 10 mm; TAPSE -1,8 cm; Right Atrium- 41\42 mm. Pulmonary Artery (PA) – 24 mm; PA Systolic Pressure - 139 mm Hg. There were no significant valvular disorders. D - deformation of the Interventricular Septum (IVS). There was a defect in the membranous part of the IVS up to 13 mm with a right to left shunt.

The Results of Main Biochemical Tests: ALT - 13 U/l, AST - 22 U/l, bilirubin - 11 µmol/l, creatinine - 36 µmol/l, lactate - 1.0 mmol/l, hemoglobin - 133 g/l. NT-proBNP - 99.52 pg/ml. According to the ultrasound of the pelvic organs the chorion has been detected on the anterior wall. The edge of the chorion was in the region of the internal so extended to 1/3 of the cervix. Fetus was in transverse presentation.

Preoperative PAH Specific Therapy Was Adjusted: it was recommended to take Sildenafil 20 mg TID. Surgical termination of pregnancy was recommended due to full chorion presentation. Additionally, temporary embolization of the uterine arteries was recommended to minimize blood loss. Epidural anesthesia with invasive monitoring, including catheterization of the radial artery and internal jugular vein, has been chosen.

Surgical procedure anesthesia CPR: 06/30/2022 09:40-10:20. The patient was prepared for surgical procedure in the cath-lab according to standard methods. 10:25. Antibacterial prophylaxis with Ampicillin + Sulbactam, (Ampicillin+Sulbactam 1,0g +0,5g; Krasfarma, Russia) 3000 mg was initiated. However, during infusion of 1000 mg of antibiotics, the patient's condition worsened (vomiting, agitation, loss of consciousness, hemodynamic depression up to 60/40 mm Hg, tachycardia up to 145 beats/min, SpO2 decreased to 56%). 10:25-10:45. Intensive therapy was started: orotracheal intubation and controlled mechanical ventilation and hemodynamic support including chest compressions. As anaphylaxis was suspected, Epinephrine bolus 100 μg was given IV, further followed by infusion 0.1 - 0.3 μg/kg/min, Infusion therapy and correction of acid-base balance were carried out, methylprednisolone 120 mg was administered. To reduce PVR and improve the hemodynamic profile, nitric oxide (NO) was inhaled at a dose of 60 ppm (synthesis from atmospheric air with he "Tianox" device, Russia). TTE and chest x-ray were performed, left ventricular injury, hemopericardium, pneumothorax and hemothorax were excluded.

ECMO Procedure: 10:45. Patient displayed arterial hypotension - 40/20 mm Hg. Due to the lack of effectiveness of CPR, the team decided to start veno-arterial ECMO. 10:45-11:30. Venous and arterial cannula implantations were performed, and an ECMO circuit was prepared. 11:30. ECMO-flow 3 l/min was initiated. It allowed to achieve stabilization of hemodynamics, an increase SaO2 up to 98%. 15:15. A spontaneous miscarriage occurred while the patient was in the ICU. The patient received Carbetocin 100 mcg to prevent bleeding. No bleeding from the genital tract was observed. The infusion of unfractionated Heparinum (UFH) required for ECMO was not carried out. 07/01/2022 00:15. Infusion of UFH 500 U/h was started. The patient underwent a course of antibiotic prophylaxis: Vancomycin 1gr BID i/v, Clindamycin 300 mg TID i/v 10:00.

The patient was extubated. 07/01/22-07/04/2022. The PAH therapy was adjusted: sildenafil (20 mg TID); bosentan (125 mg BID); selexipag (was started at dose 200 μg BID); NO inhalation at dose 40 ppm. 07/08/2022. Inhalation of NO using «Tianox» deviceis turned off. The physical activation and rehabilitation program has been upgraded. 07/11/2022.The patient was discharged from the ICU and transferred to the cardiology department. 07/29/2022 The patient was hemodynamically and clinically stable against the background of three-component PAH-specific therapy: Sildenafil (20 mg TID), Ambrizentan (10 mg OID), Bosentan (125 mg BID), Selexipag (was titrated to the maximum tolerated dose - 1600 μg BID. The patient was discharged from the Almazov center with a moderate decrease in physical status.

Discussion

We found only one publication about case of successful CPR in a pregnant woman with ES [7]. In this case, the ECMO system was available, but was not installed, since the cardiac arrest occurred due to ventricular fibrillation, which was rapidly stopped by defibrillation.



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Monday, November 30, 2020

Molecular Dynamics Simulation and Analysis of some Ligands on Var2csA Target - Juniper Publishers

 Novel Approaches in Drug Designing & Development - Juniper Publishers  

Abstract

Prevalence of malaria during pregnancy and the spate of drug resistance by malaria parasites have constantly impacted maternal, perinatal and neonatal outcomes, especially in sub-Saharan Africa. Inhibiting binding; or displacement of bound infected erythrocytes from the placenta as an adjunct treatment or vaccine for malaria was considered an option towards ending pregnancy associated malaria in this study. Molecular modeling and toxicity predictors used in this study indicated that among the ligands screened, IH3 had the lowest binding energy of -9.8Kcal/mol while var2csA had -2.8Kcal/mol. Var2csA is parasite’s adhesive protein. It was also observed that out of the 90 ligands (binding affinity range -9.8 to -1.0 Kcal/mol) screened, IH3 (-9.8Kcal/mol), FAD (-8.4 Kcal/mol), NDP (-8.2 Kcal/mol), A5A (-8.2 Kcal/mol), ABO (-8.1 Kcal/mol), IH2 (-7.8 Kcal/mol), 2RT (-7.7 Kcal/mol), CRO (-7.7 Kcal/mol) and IH1 (-7.7 Kcal/mol) appear to be the most promising lead compounds to occupy var2csA binding pocket in pCSA in order to prevent adhesion of malaria infected erythrocytes to the placenta. SwissADME and Molinspiration Cheminformatics for LogP (mean of 1.07 and range of -2.79 to 4.18) of the lead compounds showed no correlations between lipophilicity and interaction with receptors. Of all the compounds selected for analysis, only ABO and 2RT exhibited drug-like properties based on Ghose, Lipinski and Veber filters. The data therefore suggests that IH3, FAD, NDP, A5A, ABO, IH2, 2RT, CRO, IH1 and var2csA make favourable lead candidates for targeting pCSA and therefore require further in vitro and in vivo evaluations.

Keywords: Malaria; Pregnancy; Modeling; Toxicity; Auto Dock

Abbreviations: pRBCs: Parasitized Red Blood Cells; IEs: Infected Erythrocytes; DC: Domain Cassette; EPCR: Endothelial Protein C Receptors, PAM: Pregnancy Associated Malaria; DARC: Duffy Antigen Receptor for Chemokines; EC: Endothelial Cell; IPT: Intermittent Preventive Treatment; PDB: Protein Data Bank; TEST: Toxicity Estimation Software Tool; FAD: Flavin Adenine Dinucleotide

Introduction

Background to the Study

Malaria is one of the leading causes of death in tropical, underdeveloped countries throughout the world. Malaria in pregnancy is a major global public health concern; this has been attributed to transient depression in cell-mediated immunity which has increased the susceptibility of pregnant women to malaria and its adverse outcomes. Approximately 32.3% of pregnant women in sub-Saharan Africa are at the risk of malaria yearly [1]. Malaria is caused by Plasmodium sp transmitted by the bite of the female Anopheline mosquito. Upon infection, the life cycle of the Plasmodium sp. begins and ends in the bloodstream where erythrocytes are infected and consequently destroyed, causing symptoms such as anemia, respiratory sequelae, cerebral malaria, metabolic acidosis, and eventually organ failure, leading to death [2]. However, before the total destruction of erythrocytes occurs, there seems to be a mode by which the parasitized red blood cells (pRBCs) become sequestered or adhered in particular sites throughout the vascular system [3] and this results in widespread sequestration of infected erythrocytes (IEs) and hence their reduced clearance from the blood stream by the spleen [4]. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates this adhesion of IEs to various host receptors on the vascular lining during the blood stage of malaria infection [4-6].

Proteins of the PfEMP1which include var2csA (DBL2x, DBL3x and DBL6ɛ), Var1csa (DBL3γ), CIDR groups B & C, CIDR of DC8 group A, CIDR of DC13, DC4, DC8, DC5, Duffy Binding Protein (DBP) for Pv & Pk, and PfRh4 for Pf, PfRh5, EBA - 175, EBL -1, EBA - 140 and EBA 181 mediate adhesion and cell invasion through specific binding to multiple endothelial cell (EC) receptors, including domain cassette (DC) 36, DC31also known as platelet endothelial cell adhesion molecule 1 (PECAM -1) or cluster of differentiation 31 (CD31) [7], DC13, DC8, intercellular adhesion molecule -1 (ICAM - 1), E-selectin, endothelial protein C receptors (EPCR), duffy antigen receptor for chemokines (DARC), and placental chondroitin sulfate A (pCSA). Others include: CD36, complement receptor - 1 also known as CD35 (i.e.; C3b, or C4b), VLA-4, LW, CD 58, CD 147, CD44, B-CAM/LU and Glycophorin A (F actin and G actin).

In this study, we focused on pregnancy associated malaria (PAM) also referred to as placental malaria (PM) which is mediated by Plasmodium falciparum elaborated var2csA that binds to placental chondroitin sulfate A (pCSA) to bring about sequestration of malaria IEs on the placental cyncytiotrophoblast. Each Plasmodium falciparum parasite genome constitutes over 59% var genes [5,8] and individual parasite expresses a single var gene at a time [9], maintaining all other var genes in a transcriptionally silent state. Almost all members of the var family are classified into one of 3 major groups (A, B, and C) based on a combination of chromosomal location, transcription direction, and upstream promoter sequence [10]. As an important activity in many drug discoveries projects, in silico molecular modeling was employed to identify molecules with activity against pCSA and subsequent lead optimization of the hits identified [11].

Statement of the Problem

Use of insecticide treated net and intermittent preventive treatment (IPT) are currently the preventive strategies to improve maternal and fetal outcomes amidst the ravaging malaria scourge. However, due to waning efficacy of the IPTs and insecticide treated nets as a result of resistant strains of the parasite and the malaria transmitting vectors, these preventive measures have become ineffective and less reliable over-time. This problem has persisted for a while now and there is no valid vaccine yet to prevent malaria, especially, placental malaria [12].

In this study, our target is to identify ‘‘in silico”, for the purpose of clinical application, non-recombinant product(s) that can competitively inhibit Plasmodium falciparum infected erythrocytes (PfIEs) form binding, or displace bound PfIEs from placental chondroitin sulfate A since no antibody or antigen based vaccine tend to be successful in the search for vaccine against pregnancy associated malaria.

Aim and Objectives of the Study

Aim

This study aims to discover “in silico” non recombinant molecules which can interact with placental chondroitin sulfate to inhibit binding or displace bound var2csA from the placenta in order to prevent pregnancy associated malaria.

Objectives

1. To generate a library of molecules similar to var2csA (ligands to CSA) from available databases.

2. Dock these molecules against placental CSA in order to generate hits with favorable binding affinity with it; and

3. Analyze “in silico” the suitability and safety of some of the selected molecules as drugs.

Operational Definition of Terms

1. Autodock: Molecular modeling simulation software. It is especially effective for protein–ligand docking.

2. Binding affinity: The strength of the binding interaction between a single biomolecule to its ligands.

3. Databases: A collection of information that is organized so that it can be easily accessed, managed, and updated. Example here include PDB and PubChem.

4. Virtual molecular screening (also known as molecular docking): A computational technique used in drug discovery to identify those structures which are most likely to bind a drug target, typically a protein or enzyme.

Literature Review

Concept of Drug Design and Receptor Interaction

Many micro and macroscopic properties of substance, such as biological activities of drugs and drug-like compounds are controlled by their stereochemistry. Molecules with one or more rotational degree of freedom (rotatable bonds) exhibit conformational isomerism, where the stable conformers correspond to energy minima, and therefore, to the most probable structures to be found in materials, pharmaceuticals and agrochemicals [13]. Interaction stabilizing conformations are provided by certain hydrogen bonding even though others also take place in many cases, as seen in electrostatic effects and hyperconjugation phenomena, i.e, electron donation from the endocyclic oxygen lone pair toward the vicinal axial antibonding orbital [14]. For example, the core of the CSA-binding site is situated in the N-terminal part of var2csA by hydrogen bonding and van der Waal force (Figure 1). The following equation [EI ]aq [E]aq +[I ]aq is used to determine the reaction involving a receptor and its ligand. The change in free energy of binding (ΔG) is related to binding affinity, ( ΔG = −RTInkA).

Where E = Receptor, I = Ligand, KA = Equilibrium constant, R = ideal gas constant = 8.314J/mol-K, and T = Temperature of the reaction (Kelvin).

Receptor and ligand docking aim at correct prediction of the structure of the complex under equilibrium conditions. The docking techniques used in this study included prediction of the binding modes of novel drugs (ligands) and the study of proteinprotein interactions using AutoDock Vina (http://vina.scripps. edu) tool in PyRx (http://pyrx.sourceforge.net) where autodock reproduces nine different protein - ligand complexes for which the structure of the macromolecule was known without prior knowledge of the binding site. The more negative the binding affinity becomes, (i.e, the lower the binding energy), the better the orientation of the ligand in the binding pocket of the receptor.

The autodock programme package - PyRx was able to select the correct complexes based on the energy without prior knowledge of the binding site; (i.e, blind docking). PyRx was not able to see the binding site but was able to find it.

Prediction of correct structure (posing) of the [EI ] complex does not require information about kA. Prediction of biological activity (ranking) does require information about KA . For the complex [EI ] , the following factors are important: steric, electrostatic, hydrogen bonding inhibitor strain (if flexible) and enzyme (receptor) strain. Important factors when considering the equilibrium include: desolvation, rotational entropy, and translational entropy. Most scoring functions focus on energetic than entropic effects. The events of ligand-binding are driven by a combination of enthalpic and entropic effects, either of which can dominate specific interactions [15-17].

Var2csA

Var2csA is a variant of many adhesive proteins of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) elaborated on the surface of Plasmodium falciparum infected erythrocytes for the purpose of tethering infected cells on tissue receptors to evade destruction by the spleen. Var2csA protein consists of six DBL domains, a single transmembrane helix, cytopasmic acidic terminal sequence and significant interdomain regions (Figure 2). Four of the DBL domains have been shown to bind CSA [18-20].

The structure of the DBL3X domain has been shown [22,23] to have an ɑ-helical scaffold similar to those observed in DBL domains from the invasion proteins like erythrocyte-binding antigen 175 (EBA-175) and the Duffy antigen receptor for chemokines (DARC) - binding protein [24,25].

It has also been shown that full length recombinant var2csA binds specifically to CSA with nanomolar affinity, and that the CSA- binding site lies in the N-terminal part of the protein [26]. The core CSA- binding site lies within the DBL2X domain and part of the flanking interdomain regions, in contrast to the idea that single domains do not possess the structural requirement for specific CSA binding. They added that the x-ray scattering measurement revealed CSA- binding DBL2X domain is situated in the center of the structure.

Materials and Methods

Materials

The following computing resources and items were used:

Compounds: The protein data bank (PDB) structure of the receptor chondroitin sulfate A (PDB ID: 3bqk) in its raw and prepared forms is shown in Figure 4. Structures of some of the ligands used are shown in Figures 3

Hardware and Software: This work was carried out on windows 8.1 Pro with processor: Intel® Core (TM) i5 CPU M 520 @ 2.40GHz having installed memory (RAM): 4.00GB (3.86 GB usable) on system type: 64 bit operating system although 32-bit Windows Vista operating system also work well. PyRx docking software version 0.8 for Windows [14]. PyRx is open source software to perform virtual screening. It is a combination of several softwares such as AutoDock Vina, AutoDock 4.2, Mayavi, Open Babel, etc.

PyRx uses Vina and AutoDock 4.2 as docking softwares. In this study, AutoDock Vina [27] was used. Discovery studio (Discovery Studio: v20.1.0.19295), Pymol (Pymol stereo 3D quad buffer) and ICM Browser (Molsoft MolBrowser 3.8-7d) were used to examine structural properties and study binding interaction between receptor residues and ligands. SwisADMET, and Molinspiration cheminformatics were used for evaluation of drug-likeness of the ligands and Toxtree [28] was additionally used to determine the toxicity and safety profile of the ligands.

Databases and Applications: Protein data bank (rcsb.org) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) were used to download the chemical structures of the receptor (3bqk) and those of the ligands. Canonical SMILES and other information about the ligands and the receptor were extracted from PubChem. Toxtree, Toxicity Estimation Software Tool (TEST), SwissADMET, Molinspiration and Lazar Toxicity Predicter were employed to test various toxicity and safety parameters of lead compounds.

Methods

Retrieval of Macromolecule: The structure of the macromolecule-chondroitin sulfate A (CSA) was retrieved by searching in the protein data bank (PDB) (rcsb.org/ structure/3bqk); downloaded and saved as a PDB format. Retrieval of Liands: Molecules constituting the ligands screened belong to the same enzyme commission (EC) class of CSA and were downloaded from rcsb.org as Excel file. In the file, the ID values of all the class members were copied and pasted on a page “download ligand” site in the PDB in order to generate their corresponding ideal structure data file (SDF) formats. The “launch download” application of PDB downloaded and channeled the ligands into a folder (ligands) that was created on the desktop. Some of the ligands are presented in Table 1.

Target Preparation and Docking Process: The x-ray structure of the receptor (3bqk) was downloaded from protein data bank (rcsb.org), cleaned from hetatoms in discovery studio and resaved (Figure 4). The input ligand files were also prepared for virtual screening by converting them to PDBQT file format when they were imported into PyRx software as chemical table file (SDF). Following a series of steps, the library of the ligands was docked into the active site of 3bqk in the PyRx platform using Amber Van der Waals in the active box of 17 x 15 x 17. Autodock Vina took each ligand and bonded its different conformations to the receptor molecule (3bqk) to get the binding energies in different orientations of each ligand. Each ligand has nine different binding orientations starting from 0 to 8.

Validation of Docking Process: Docking was repeated three times on the same system specifications for the purpose of process validation and all returned minimal variation (P<0.01 data not shown) in uff energy, binding energy and RMSD values.

Binding Analysis: Discovery studio (Discovery Studio: v20.1.0.19295), Pymol (Pymol stereo 3D quad buffer) and ICM Browser (Molsoft MolBrowser 3.8-7d) were all used to analyze binding interactions between residues of 3bqk and the ligands.

Drug likeness: Toxtree, Toxicity Estimation Software Tool (TEST), SwissADMET, Molinspiration and lazar toxicity predictions were variously employed to test toxicity and safety parameters of lead compounds.

Results and Discussion

Results

Analysis of Docking Result: A library of 90 compounds in the same EC class with chondroitin sulfate A, known to interact with pCSA was generated from rcsb.org. Compounds in the library demonstrated good binding affinity with many having higher binding affinity to pCSA (3bqk) than 2y8d a var2csA molecule and SO4, a functional species in chondroitin sulfate A (Table 2).

The first 75 ligands and SO4 have binding affinity higher than that of 2Y8D. This implies that var2csA can be displaced from pCSA in preference for molecules which have higher binding affinity. 2Y8D and SO4 which have their highest binding affinity as-2.8Kcal/mol and -3.7Kcal/mol also ranked 719th and 655th respectively. For this study, only the first nine ligands with the highest binding affinity (-9.8 to-7.7Kcal/mol), 2y8d and SO4 were selected for the target analysis. The complex formed for some of the ligands are represented in figure 5, the complexes have different structural modification from the SDF format seen in Figure 4 except for SO4 which it is not a valid selection for binding analysis. It is not a chain, molecule or a multiple residue object.

Analysis of Binding Interactions: Receptor residues interacting with SO4, 2y8d and the top nine selected ligands as seen in Discovery studio and Pymol are listed in Table 3a.

Evaluation of Drug likeness and Organ Toxicity of some Selected Hits: The lipophilicity for selected compounds is as shown by the values of their consensus logP in Table 4. Drug likeness of the compound’s Tables 5a & 5b.

Discussion

All the ligands in this study yielded their lowest binding energy at their first binding modes (0). The reliability of this study was ascertained by triple - docking of the ligands with the receptor (pCSA) and it was found that both the first, second and the third dockings maintained the same values in all parameters and for all binding modes (P < 0.01 data not shown), giving IH3 and FAD as the first two ligands with the lowest binding energy as shown in table 2 and implicitly, the ligands of choice to bind placental chondroitin sulfate during in vitro and in vivo evaluations. The binding affinities of the top nine compounds on var2csA target are closely related that they lie within a close range of -9.8 to -7.7Kcal/ mol indicating that their potential as lead compounds for 3bqk is comparable. Auto Dock reproduces nine different protein -

ligand complexes for which the structure of the macromolecule was known, without prior knowledge of the binding site but here only the result from the first binding orientation was presented since it is the one with the highest binding affinity, the full binding orientations and their RMSD values for all the ligands are available on request. The RMSD for the first binding orientation is always 0. The binding affinity and the RMSD value collectively determine the ranking of the molecules.

Analysis of Binding Pattern

Considering their binding interactions, FAD had the highest number of favorable hydrogen bonds with receptor residues but shares equal number of hydrophobic contacts with IH3 (Table 3a & Table 3b). FAD is a drug already in used to treat eye diseases caused by vitamin B2 deficiency, such as keratitis and blepharitis (Drugbank, http://www.drugbank.ca/). Some other compounds like NDP, ABO, IH2, CRO, IH1 and SO4 possess no hydrogen bonds but have good number of other hydrophobic contacts except CRO and SO4 which do not have any significant favourable bonds with receptor residues based on observation from Discovery Studio (Figures 6).

Drugability of Compounds

No correlation was observed between lipophilicity and the interaction with the receptor by considering cLogP values and number of conventional bonds (Tables 3b & Table 4). However, for FAD, NDP and A5A, interaction with the receptor is correlated with low lipophilicity while 2RT has high cLogP value. The compounds with high cLogP values used their polar functional groups to interact with the receptor. FAD, 2Y8D and IH3 exhibited good hydrogen bond interaction with var2csA receptor of placental chondroitin sulfate A possibly due to their polarity.

Screening the compounds for drug likeness using Lipinski, Veber and Ghose filters, FAD, A5A and IH1 did not satisfy the requirements for a safe drug in all the filters used. However, only ABO and 2RT suitably satisfied the conditions for drug likeness in all the filters and therefore likely the safest (Table 5b). Notwithstanding these observations, all the compounds selected can be relatively safe since the unsatisfied molecules tend to violate 1 or 2 conditions in 1 or 2 of the three filters. Again, FAD which violated almost all the conditions in all the three filters is already in the market for other medical uses.

Based on toxicity prediction of some compounds in Lazar Toxicity Predicter, FAD, NDP and ABO penetrate the human blood brain barrier (Table 6) indicating possibility of central nervous system toxicity. But this could also be an advantage for the compound to access the brain to elicit their pharmacologic activity in the event of susceptible cerebral malaria. They also tend to be non-carcinogenic in mouse model based on observations from the same platform.

Summary of Findings, Conclusion, and Recommendations

Summary of Findings

In this study, an extensive search was carried out in silico to discover non recombinant substances that could serve as vaccines or adjunct treatment for pregnancy associated malaria (PAM) also known as placental malaria (PM). Ligands (or small molecules or drugs) with activity against chondroitin sulfate were extracted from designated drugs and proteins databases. The ligands were screened against a receptor (pCSA, PDB ID: 3BQK) to isolate molecules that can favourably bind it thus displacing or inhibiting binding of var2csA, a Plasmodium falciparum (P.f) protein usually deposited on the surface of P.f- infected erythrocytes that enhances the adhesion of the parasite carrying cells to the endothelium of tissue vasculature, thereby tethering them away from splenic destruction.

Ninety ligands were screened out of which 75 were found to have higher binding affinity with pCSA than var2csA, with binding affinity ranging from -9.8 to -2.8 (Table 2, complete data not shown). Out of this number, the best nine ligands (IH3, FAD, NDP, A5A, ABO, IH2, 2RT, CRO and IH1,) whose binding affinities ranged from -9.8 to -7.7 were isolated for further in silico evaluations. Their analysis showed that four compounds (IH3, FAD, A5A and 2RT) had hydrogen (strong covalent) bond interaction with 3bqk while five (NDP, ABO, IH2, CRO and IH1) had no hydrogen bond with 3bqk. One compound (CRO) does not have both hydrogen and hydrophobic bonds with 3bqk, yet it has high binding affinity with the receptor. This was possible because, the compound may have used its polar functional groups to make the interaction. The same is applicable to SO4 (a sulfate ion) which also do not have any hydrogen or hydrophobic contact with the receptor. 2Y8D had both hydrogen bond and hydrophobic bond interactions with the receptor (Table 3a & Table 3b).

In silico drug ability testing of the 9 selected compounds including SO4 and 2y8d indicates that only four compounds (ABO, 2RT, CRO, and 2Y8D) based on Lipinsky, Veber and Ghose rules perfectly satisfied drug likeness conditions (Table 5b). That means 2Y8D can be administered to mimic the parasitic form of it in binding placental chondroitin sulfate A. FAD violated all the rules for a safe drug but it has been approved for use in the treatment of eye diseases caused by vitamin B2 deficiency, such as keratitis and blepharitis. Lazar Toxicity Predicter (Table 6) showed FAD, NDP and ABO to have ability to cross blood brain barrier and non-carcinogenic.

Conclusion

Following the analysis and observations so far, all the 9 selected compounds including SO4, 2y8d and all the rest of the compounds screened deserve further in vitro and in vivo evaluations to determine which compound has the best in vivo activity against placental malaria and safe for human use.


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