Monthly Archives: April 2024

Prof. Subhash Mukhopadhyay: The doctor behind the first Indian life outside the womb

The brilliant doctor who facilitated the birth of India’s first ‘test tube’ baby as a result of his original, ingenious research, was forced to take his own life due to offensive and active apathy of the then Left Front government in West Bengal.

The year 1978, separated by 67 days, saw the birth of two miracle babies, Louise Brown and Durga (Kanupriya Agarwal). Both were the first babies produced using a new medical technology called in vitro fertilization (IVF), commonly known as test tube babies. Though the description of similar methods has been in ancient Indian textbooks, the birth of both babies was a breakthrough in the modern medical sciences that has become a boon for infertile couples globally. Two great doctors working independently at the same time 8,000 kilometers apart achieved this feat. Prof. Robert G. Edwards, the doctor behind the birth of Louise Brown, became famous for his path-breaking work, and he established the world’s first clinic for IVF therapy at Cambridge called Bourn Hall Clinic. He was awarded Nobel Prize in Medicine in 2010. In contrast, the brainchild behind Durga, Prof. Subhash Mukhopadhyay, who achieved the same feat, was denied recognition and faced the hostility of the CPI(M) led Left Front government in West Bengal, which had rubbished his research. The government ridiculed his work, and he was ostracized, which pushed him to end his life. Ironically, the doctor who created the first Indian life outside the womb ended his own life due to banishment and torture by the communist government. His tragic life story has inspired the novel Abhimanyu (by Ramapada Chowdhury) and the famous Bollywood movie Ek Doctor Ki Maut.

Development of novel methods for in vitro fertilization (IVF)

Fertilization is the first process during pregnancy when the egg and sperm fuse at the ampulla of the fallopian tube, forming a zygote. The germinal stage of embryonic development begins at the zygote, which undergoes mitotic cell divisions and later transplants as a fetus at the uterus after nine weeks of fertilization. The fetus undergoes further development for another 31 weeks, and then a child is born.

Many paternal and maternal ailments cause the impairment of the natural process of fertilization. To overcome this, many researchers, including Prof. Robert G. Edwards and Dr. Patrick Steptoe at Cambridge, started working on harvesting eggs and sperm, fertilizing them outside the human body, and then transferring the zygote back to the uterus. At the same time, Prof. Subhash Mukhopadhyay, who was a Professor of Physiology at Bankura Sammilani Medical College, formed a team with Prof. Sunit Mukherjee, a Professor of Food Technology & Biochemical Engineering at Jadavpur University and Dr. Saroj Kranti Bhattacharya, Associate Professor of Gynecology & Obstetrics at Calcutta Medical College to develop a method for successful in vitro (Latin word meaning ‘in glass’) fertilization. Prof. Mukhopadhyay stimulated the ovary using human menopausal gonadotropin (hMG) (also called Menotropin) to increase the success probability. Since Prof. Edwards’s team failed to achieve stimulation, it used a retrieved oocyte from a natural mensural cycle for fertilization. To overcome the problem of a shortened luteal phase, which was one of the reasons for the failure of stimulation, Prof. Mukhopadhyay developed the cryopreservation technique for human embryos. He cryopreserved the human embryo using DMSO as a cryoprotectant before transferring them, after thawing, in another natural cycle. In contrast to the Cambridge team’s invasive trans-abdominal laparoscopic procedure, Prof. Mukhopadhyay employed a new transvaginal colpotomy procedure to fetch the oocytes, which was a minimally invasive and more efficient method.

Ovarian stimulation using hMG hormone, a minimally invasive transvaginal approach for aspirating oocytes, and cryopreservation of embryos, the three major ingredients of IVF developed by Prof. Mukhopadhyay, are currently the standard practice used by IVF clinics across the globe.

The Durga story

Mr. Prabhat Kumar Agarwal and Mrs. Bela Agarwal approached Prof. Mukhopadhyay for infertility treatment, which was primarily due to an ailment in the fallopian tubes. Though cautioned by Prof. Mukhopadhyay that the procedure could result in the deformation of the baby, the couple agreed to “try a new method.” He performed ovarian stimulation using hMG and aspirated fluids from follicles. After screening, he co-incubated them with the sperm for 24 hours in flasks for fertilization. The resulting zygotes developed into embryos which he slowly froze in another flask before cryopreserving them. After 53 days of cryopreservation, he thawed the embryos and transferred them to the uterus of Mrs. Bela Agarwal in her later mensural cycle. He performed all the procedures at his home, maintaining appropriate conditions. She delivered a healthy baby on 3rd Oct 1978 through a caesarian procedure. The news was made public by Amrita Bazar Patrika on 6th Oct 1978. In the report, Amrita Bazar Patrika quoted Dr. Mani Chhetri, the Director of Health Services (DHS), as finding the claim “quite convincing. If the team could now prove it, West Bengal would get a place of pride in the medical world”. Since she was born on the first day of Shardiya Navratri that year, she was named Durga. Later the parents changed her name from Durga to Kanupriya before her school admission as they feared their daughter could be treated as abnormal.

Prof. Subhash Mukhopadhyay presented his pioneering work at various conferences in India and published the cryopreservation methodology in the Indian Journal of Cryogenics in 1979.

Ignominy and tragedy

The path-breaking work of Prof. Subhash Mukhopadhyay, resulting in the birth of the first Indian IVF baby, was derided by the Indian medical fraternity. At a meeting organized by the Indian Medical Association (IMA) and the Bengal Obstetrics and Gynaecological Society (BOGS) at Chittaranjan Cancer Institute, he presented pictures of in vitro embryos, but the people ridiculed him. The DHS, West Bengal, barred him from presenting his work at any conference and refused permission to apply for a passport to attend international conferences to which he was invited. The Left Front government in West Bengal set up a four-member inquiry committee under the chairmanship of Dr. Mrinal K. Dasgupta, a Radio-Astronomer, in which neither he nor other members had the required expertise to evaluate the work.  The committee not only doubted every aspect of his work but put ridiculous farrago of infuriating cretinous questions to humiliate him. He said to the committee, ‘It is fine, don’t believe me, I will do it again. That is how science works.’ Fearing the same fate, the parents of Durga refused to participate in the inquiry or undergo any medical checkup. The committee, in its four-page report submitted to Shri. Nani Bhattacharjee, Health Minister in the Jyoti Basu government, concluded the work to be bogus and unfeasible. Following this, the government transferred him to the Regional Institute of Ophthalmology, and he was not allowed to pursue his work.

He suffered a heart attack due to stress in 1980. Facing ignominy, he committed suicide by hanging on 19th June 1981. In his suicide note, he wrote, “I can’t wait every day for a heart attack to kill me.” A brilliant scientist who could have contributed immensely to the field of reproductive biology with his cutting-edge research and brought laurels to India, he left the world dejected and unrecognized.

Lifting the Veil on the Doctor’s Work

Although his wife, Mrs. Namita Mukhopadhyay, and colleague Prof Sunit Mukherjee continued their relentless effort to get him due recognition, he and his work slowly faded into oblivion.  Two decades later,  Dr. T.C. Anand Kumar, ex-Director ICMR-NIRRH, credited with the birth of Harsha Vardhan Reddy Buri, India’s first “scientifically documented” IVF baby on 6th August 1986, lifted the veil on the work of Prof. Subhash Mukhopadhyay. Like a true scientist, during his visit to Kolkata in 1997 to attend Indian Science Congress, Dr. Anand Kumar went through the documents of Prof. Mukhopadhyay, which convinced him that Prof. Subhash Mukhopadhyay was the creator of the first Indian test tube baby. He publicly acknowledged this and wrote an article in different journals. Had he kept silent world would have never known the accomplishments of Prof. Mukhopadhyay.

In 2002 Indian Council of Medical Research (ICMR) recognized Prof. Subhash Mukhopadhyay as the pioneer of IVF in India and later started an award in his memory in 2012. At an event organized in memory of Prof. Mukhopadhyay in 2003, Durga also came forward and narrated the story behind her birth. In 2007 Prof. Mukhopadhyay was included in the Dictionary of Medical Biography, UK. After Ronald Ross and U. N. Brahmachary, he became the third scientist from Kolkata to be included in the list. The Department of Reproductive Physiology at Nil Ratan Sircar Medical College was later renamed after him. “Dr. Subhas Mukherjee Memorial Reproductive Biology Research Centre” at Behala, Calcutta, was established in 1985 by the Jadavpur University, Indian Cryogenics Council, and Behala Balananda Brahmachari Hospital. In 2018 his life-sized statue was unveiled at his birthplace Hazaribagh.

Other notable contributions

He was the first to observe a correlation between emotional stress and PCOD that causes infertility. He discovered that the hCG hormone is important for maintaining the corpus luteum immediately after fertilization and is required for healthy menstrual cycles. He also contributed significantly to understanding Testicular Feminization Syndrome and advocated using Fish Protein Concentrate (FPC) as a nutritional supplement.

Biography

Prof. Subhash Mukhopadhyay was born to Dr. Satyendra Nath Mukherjee, a famous radiologist, and Mrs. Jyotsna Devi at Hazaribagh, Bihar (now Jharkhand) on 16th January 1931. He was a descendant of Krittibas Ojha, who had composed Krittivasi Ramayan in Bengali.  After schooling at Calcutta, he graduated with honors in Physiology from the Presidency College, Calcutta. He received his MBBS degree at the National Medical College, University of Calcutta, in 1954. He was awarded the ‘Hemangini’ Scholarship and College Medal for securing the first rank in Obstetrics and Gynecology. In pursuit of his keen research interest, he worked on “The biochemical changes in normal and abnormal pregnancy” for his first Ph.D. in Physiology under the guidance of Dr. Sachchidananda Banerjee at the Presidency College, Calcutta. In 1961 he received Colombo Scholarship to work at the Clinical Endocrinology Research Unit in Edinburgh. Under the tutelage of Prof. John A. Loraine, he worked on “Developing new, sensitive bioassays for luteinizing hormone based on depletion of cholesterol in rat ovaries,” leading to his second Ph.D. degree. He returned to India in 1967 and joined Sir Nil Ratan Sarkar Medical College, Calcutta, as a Lecturer, where he later became Professor. Staying at the college campus, he built an animal house for research purposes that housed animals ranging from murine to primates. The government transferred him as a professor of Physiology at Bankura Sammilani Medical College, where he later became head of the department and worked on IVF leading to the birth of Durga. He married Namita in 1960, and they remained a child-free couple by choice as he wanted to complete his research.  

G. N. Ramachandran: A genius who laid the foundation of protein structures and 3D medical imaging

If you think you know it, then you do not know it, and if you know that you cannot know it, then you know it.

In one of his Mathematical Philosophy (MATPHIL) reports, G. N. Ramachandran elaborated on this interesting paradox from Kena Upanishad, describing the Divine force of the Universe, which conveys perpetual doubt and indefiniteness.

Prof. Gopalasamudram Narayana Ramachandran, affectionately known as GNR, was the finest molecular biologist whose seminal work in structural biology had put India on the world map of modern biology. The structural Biology era started with Linus Pauling and his colleague’s discovery of the  -helical structures of polypeptides, which set the stage for our present-day understanding of protein function. The term molecular biology was coined in 1938 by Warren Weaver, a mathematician, and Director of the Natural Sciences Division at the Rockefeller Foundation, defining an amalgam of  Physics, Chemistry, and Biology. Still, the molecular biology revolution started with the discovery of the double-helical structure of DNA by Rosalind Franklin, Francis Crick, James Watson, and Maurice Wilkins in the early 1950s. During that period, most of the work in molecular biology was done in UK and USA. Following this, a new structural fold comprising a coiled-coil triple helix was described by GNR in a series of papers published between 1954 and 1956 in Nature with his student Gopinath Kartha. Later in the 1950s and 1960s, he applied stereochemistry principles to generate a two-dimensional plot that accurately described allowed conformations of proteins. The map, now famously known as the Ramachandran plot, has become an inalienable part of molecular biology and biochemistry textbooks.

Passionate X-ray crystallographer

After graduating from St. Joseph’s College, Trichy, in 1939, Ramachandran joined the Electrical Engineering department at the Indian Institute of Science (IISc), Bangalore, and later moved to the Physics department. GNR studied crystallography all by himself while working for D.Sc. degree under the supervision of Sir. C. V. Raman. While transferring GNR to the Physics department from the Electrical Engineering department at the IISc, Raman, who was the Director of IISc at that time, said, “I am admitting Ramachandran into my department as he is a bit too bright to be in yours…”.  After completing his doctorate, GNR moved to Cavendish Laboratory, Cambridge, where he worked on mathematical theory to determine elastic constants of cubic crystals from diffuse X-ray diffraction and obtained his second doctorate. After returning from Cambridge in 1949, he joined as an assistant professor at IISc, where he nurtured the X-ray diffraction laboratory. At the initiative of Dr. A. Lakshmanaswamy Mudaliar, the  Vice Chancellor of Madras University at that time, GNR joined Madras University in 1952 as a founding member of the Physics department. Ramachandran started cutting-edge research in crystallography that included phase determination when anomalous dispersion is present, the probability distribution of X-ray intensities, crystallographic statistics, and so forth. He derived the correct formula to calculate the X-ray phase angles using Bijvoet differences, which occur during anomalous X-ray scattering. In 1971 he returned to IISc and established the Molecular Biophysics Unit (MBU), now a leading center of research in structural biology.

The Triple helix

It has been known for a long time that form and shape of all mammals are dependent on collagen protein which makes up a third of all proteins in their bodies. Collagen is vital for structural integrity; imperfection or instability in its structure causes many disease conditions. Ramachandran and Kartha used X-ray diffraction data on the collagen derived from the Kangaroo tail tendon to delineate its structure. The structural model had three parallel left-handed helical polypeptide chains, side-by-side coiled coils packed together in a hexagonal array. The triple helical structure of collagen has been described as “braided in the manner of the pigtail of a long-haired maiden from Madras.” The collagen structure was alluding to the structural biologist of that time, and the work of GNR put India on the world map of structural biology. Ramachandran mentioned that he got the idea of a triple-helix coiled coil model from the rotation and revolution motion of the moon. 

Despite his ground-breaking research, recognition did not come easily to GNR. Other leading structural biologists of that time, like Alexander Rich and Francis Crick, who were also working on collagen structure, did not accept the model citing steric issues and discrepancies in number of inter-chain hydrogen bonds. As collagen contains hydroxyproline residues GNR in his model had more than one hydrogen bond, which were mediated by water molecule. Now the controversy is well settled in favor of the ‘Madras model’ of Ramachandran after the high-resolution crystal structure of collagen (PDB id 4OY5 [0.89 Ã…]) showed that there are, on average, 1.5 hydrogen bonds in the structure. Credit for collagen structure eluded him during his lifetime, but now the record has been set straight in favor of GNR. The basic three-dimensional structure of collagen unraveled by GNR helped us understand the molecular basis of its function and diseases caused by changes in its native structure.  

The controversy on the collagen structure based on steric issues made GNR more determined, and he started working on formulating a general stereochemistry rule for protein structure that resulted in Ramachandran Plot, which has immensely benefitted protein science and drug discovery research.

Ramachandran plot

Proteins are made of twenty naturally occurring amino acids joined together like pearls in a necklace. It is called its primary structure, where amino acid residues are joined by rigid planer peptide bonds. This linear protein chain further folds into secondary (a-helix and b-strand), tertiary and quaternary structures acquiring a unique three-dimensional shape that dictates its function. Understanding a protein’s function or any dysfunction leading to disease conditions necessarily requires knowledge of its three-dimensional structure.

Ramachandran working with Ramakrishnan and Sasisekharan analyzed all the crystal structures of amino acids and observed that nonbonded atoms were closer to each other than the sum of their respective van der Waals radii. Using this and Pauling’s a-helix information, GNR used mathematical calculation (without computers…!!!) to arrive at allowed rotations of rigid planer peptide planes that will lead to sterically acceptable contacts between them. The 2D plot of these pair of allowed angles, Phi-Psi (f y), gave birth to the Ramachandran plot. The Ramachandran plot was proved correct later when the first crystal structure of a protein was determined. The plot remains valid today when around two lakh protein structures have been experimentally determined and their coordinates deposited in the Protein Data Bank (PDB). For accuracy and acceptability, all experimentally determined structures of proteins as well as predicted structures, must confer to this map. Like the Raman effect, Bose-Einstein statistics, and Chandrasekhar limit, the Ramachandran plot has immortalized the scientist behind it. 

Describing Ramachandran plot Prof. Dame Janet Maureen Thornton, a senior scientist and director emeritus at the European Bioinformatics Institute(EBI), EMBL, had written, “It never fails to excite me, when I see the Ramachandran plot and realize how much of the beauty and order of protein structures is encapsulated by this plot. I also think that this major discovery highlights the importance of clear thought and vision that do not always need expensive equipment and huge teams of people”.

Medical Imaging

G N Ramachandran (GNR) also made seminal contributions that made a deep impact to the field of three-dimensional medical imaging. In 1971 he published two exciting research papers on Projection Reconstruction; one titled ‘Reconstruction of substance from shadow: 1. Mathematical theory with application to three-dimensional radiography and electron micrography’. In another publication co-authored with A. V. Lakshminarayanan published in the same year in Proceedings of National Academy of Sciences, USA, he suggested using convolutions in the spatial dimension. Both opened the window for creating three-dimensional (3D) images of human anatomy using two-dimensional (2D) images, which could provide depth information. This led to the development of the Computed Tomography scan (CT-scan), where several 2D X-ray images obtained at different angles and depths, called projections, are used to construct 3D images on a computer.

Magnetic Resonance Imaging (MRI) is another imaging technique that uses a projection reconstruction algorithm developed by Ramachandran. In the first publication describing MRI in Nature in 1973, Paul Lauterbur cited Ramachandran’s algorithm as a method for projection reconstruction. Lauterbur, who received Nobel Prize in Physiology in 2003, had initially called the new imaging technique zeugmatography derived from the Greek word zeugma meaning “that which is used for joining.” MRI uses linear gradient in a homogeneous magnetic field of a Nuclear Magnetic Resonance (NMR) spectrometer so that different points in the object experience a slightly different magnetic field. This leads to the NMR spectrum as a projection of the 2D object into a 1D spectrum. In MRI, several projections are acquired, and images are reconstructed using the projection reconstruction algorithm of Ramachandran.

Both CT-scan and MRI have benefited enormously from G N Ramachandran’s 1971 papers, and he is rightly credited for having a deep impact on the ‘Medical Imaging’ field. In a nutshell, Ramachandran’s contribution has revolutionized human radiology helping with a high-throughput, efficient, and accurate diagnosis, which has become a boon for the modern medical sciences.  

Personal life and Vedanta

G N Ramachandran was born on October 8,1922 in Ernakulam in Kerala. He was the eldest son of G.R. Narayana lyer and Lakshmi Ammal. His father was a Professor of mathematics who taught him mathematics.  After retiring from MBU, he continued as a Professor of Mathematical Philosophy at IISc until 1989. In 1999 GNR was awarded the 5th Ewald Prize by the International Union of Crystallography for his outstanding contributions in crystallography. He spent his final years in Chennai, where he passed away on April 7, 2001.

Syadvada and Saptabhangi of Jain philosophy had a profound impact on Ramachandran. In one of his papers published in the journal Current Science in 1982, he described their usage in Boolean Algebra. In addition to applying mathematics to biological problems, Ramachandran used mathematics to look into divinity.  In ‘Vedanta and Epistemology’ he wrote, “There is a close analogy of these ideas of modern logical analysis with the concepts in Indian philosophy of the ‘Infinite’ (Ananta), which is one of the attributes of Brahman (Absolute Reality). The Upanishads contain many statements to the effect that this Reality has contradictory properties … The logical similarity to the example of infinity in mathematics is very close. In the case of mathematical infinity, we have to say that although it is a number, it does not belong to the class of finite numbers, and therefore it can have contradictory properties such as being both greater than and smaller than itself.”

Though his contributions like the Collagen triple-helix structure; Ramachandran plot; and Tomography should have got not one or two but three Nobel prizes, he remains a shining jewel missing in the Nobel crown.